Kunxinning and preparation method and application thereof

Abstract

The invention belongs to the field of traditional Chinese medicine, and particularly relates to Kunxinning and a preparation method and application thereof. The effective components of the Kunxingning disclosed by the invention are artemisinin and artemisinin. Animal experiments prove that kunxingning can improve the early-onset ovarian insufficiency toxicity state of SD rats caused by tripterygium glycosides, reduce ovarian granular cell apoptosis, improve the ovarian pathological state, promote follicular development, restore the serum sex hormone level to be normal to a certain extent, improve the state of proportion imbalance of Th17 / Treg immune cells, and improve the early-onset ovarian insufficiency toxicity of the SD rats. And the mood cycle of the rats is recovered to a certain extent. Meanwhile, the physiological activity of the tripterygium glycosides composition obtained by compounding the kunxingning and the TG is equivalent to that of the TG.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and particularly relates to Kunxining, a preparation method and application thereof. Background Art

[0002] Tripterygium glycosides (TG) are a mixture of highly polar, fat-soluble components extracted and purified from the dried, peeled root core of Tripterygium wilfordii (Tripterygium wilfordii Hook.f.), a plant of the Celastraceae family. Its physiological activity is synergistically produced by diterpenoids such as triptolide and triptolide ketone, triterpenoids such as triptolide and triptolide A, and alkaloids such as triptolide and triptolide. TG, known as a "Chinese herbal hormone," is currently used clinically as a nonsteroidal immunosuppressant for the treatment of rheumatic and autoimmune diseases such as nephrotic syndrome, Behçet's disease, systemic lupus erythematosus, Crohn's disease, and rheumatoid arthritis. It also has promising clinical therapeutic effects on a variety of inflammatory diseases, including chronic gastritis, ankylosing spondylitis, optic neuritis, and urinary tract disorders.

[0003] However, TG preparations also exhibit significant toxic side effects, severely damaging the reproductive, digestive, liver, kidneys, hematopoietic, immune, nervous, and urinary systems. Therefore, reducing these toxic side effects remains a challenge in clinical practice. Currently, combined medications are commonly used to attenuate the toxicity of TG, significantly reducing its liver damage. However, for attenuating the reproductive system, particularly the female reproductive system, an ideal TG-based combination drug that does not affect its physiological activity while effectively reducing its toxicity to the ovaries remains elusive. Summary of the Invention

[0004] According to one aspect of the present invention, Kunxining is provided. Kunxining is an Artemisia annua extract with low ovarian toxicity, and its active ingredients are artemisinin and artemisinin.

[0005] In some embodiments, Kunxining is an Artemisia annua extract rich in artemisinin and artemisinin, wherein the mass ratio of artemisinin and artemisinin in the Artemisia annua extract rich in artemisinin and artemisinin is (0.8-1.2):(0.8-1.2), and the mass ratio of artemisinin and artemisinin to the Artemisia annua extract rich in artemisinin and artemisinin is 85-95%.

[0006] In some embodiments, the mass ratio of artemisinin to artemisinin in the Artemisia annua extract rich in artemisinin and artemisinin is 1:1, and artemisinin and artemisinin account for 90% of the mass ratio of the Artemisia annua extract rich in artemisinin and artemisinin.

[0007] According to a second aspect of the present invention, there is provided the use of Kunxining in preparing an Artemisia annua preparation.

[0008] According to a third aspect of the present invention, there is provided a use of Kunxining in the preparation of a medicament for reducing the toxicity of tripterygium wilfordii glycosides to the female reproductive system.

[0009] According to a fourth aspect of the present invention, there is provided a use of Kunxining in the preparation of a drug for reducing the ovarian toxicity of tripterygium wilfordii glycosides.

[0010] The beneficial effects of the present invention include:

[0011] Animal experiments have demonstrated that Kunxining can improve the toxicity of premature ovarian insufficiency caused by triglycerides (TG) in SD rats, reduce ovarian granulosa cell apoptosis, improve ovarian pathology, promote follicular development, restore serum sex hormone levels to normal to a certain extent, improve the imbalance in the Th17 / Treg immune cell ratio, and restore the rat estrous cycle to a certain extent. Furthermore, the physiological activity of the tripterygium wilfordii polyglycosides composition obtained by combining Kunxining with TG is comparable to that of TG. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The fitted standard curve and linear equation were obtained from the liquid phase data of pure artemisinin.

[0013] Figure 2 This is the HPLC spectrum of the high-content artemisinin component.

[0014] Figure 3 This is the HPLC spectrum of Kunxining.

[0015] Figure 4 The diagram shows the changes in the ovaries and uteri of rats in each group. Note: Figure 4 A. Anatomical diagram of the ovary-fallopian tube-uterus of rats in each group, Figure 4 B. Ovarian index of rats in each group, Figure 4 C. Uterine index of rats in each group. **: P < 0.01 compared with the Con group; ##: P < 0.01 compared with the TG group.

[0016] Figure 5 The effect of AE-n1 on the estrous cycle of TG-induced POI rats (M (P25-P75), n=8). Note: **: compared with the Con group, P < 0.01; #: compared with the TG group, P < 0.05; ##: compared with the TG group, P < 0.01.

[0017] Figure 6 Effects of AE-n1 on serum sex hormone levels in TG-induced POI rats ( n=8), Note: Figure 6 A. Serum LH levels of rats in each group; Figure 6 B. Serum FSH levels of rats in each group; Figure 6 C. Serum E2 levels of rats in each group; Figure 6 D. Serum AMH levels of rats in each group. **: P < 0.01 compared with the Con group; #: P < 0.05 compared with the TG group; ##: P < 0.01 compared with the TG group.

[0018] Figure 7 The effect of AE-n1 on the ovarian tissue morphology of TG-induced POI rats (200×). Note: Figure 7 A. HE staining results of ovarian tissue of rats in Con group; Figure 7 B. HE staining results of ovarian tissue of rats in TG group; Figure 7 C. HE staining results of ovarian tissue of rats in AE-n1-L group; Figure 7 D. HE staining results of ovarian tissue of rats in AE-n1-M group; Figure 7 E. HE staining results of ovarian tissue of rats in AE-n1-H group; Figure 7 F. HE staining results of ovarian tissue of rats in EV group.

[0019] Figure 8 The effects of AE-n1 on ovarian follicles at all levels in TG-induced POI rats (M (P25-P75), n=8). Note: *: P < 0.05 compared with the Con group; **: P < 0.01 compared with the Con group; #: P < 0.05 compared with the TG group; ##: P < 0.01 compared with the TG group.

[0020] Figure 9 The effect of AE-n1 on the ultrastructure of ovary in rats with POI induced by TG (5000×-30000×).

[0021] Figure 10 Effect of AE-n1 on apoptosis of ovarian granulosa cells in rats with POI induced by TG (200×)( n=8), Note: Figure 10A .TUNEL expression in granulosa cells of ovarian follicles in rats of each group (200×); Figure 10B Statistical graph of the mean fluorescence intensity of TUNEL in granulosa cells in the ovarian tissue of rats in each group. **: P < 0.01 compared with the Con group; ##: P < 0.01 compared with the TG group.

[0022] Figure 11 The effect of AE-n1 on the expression of apoptosis-related proteins in ovarian apoptosis rats induced by TG (x±s, n=8). Note: Figure 11A Changes in TNF-R1 protein expression in the ovaries of each group; Figure 11B .Changes in ovarian Cyto-C protein expression in each group; Figure 11C.Changes in Apaf-1 protein expression in the ovaries of each group; Figure 11D Changes in XIAP protein expression in the ovaries of each group; Figure 11E .Changes in the expression of Bax and Bcl-2 proteins in the ovaries of each group; Figure 11F .Changes in ovarian Bax protein expression in each group; Figure 11G .Changes in Bcl-2 protein expression in the ovaries of each group; Figure 11H .Changes in the Bax / Bcl-2 ratio in the ovaries of each group; Figure 11I Changes in ovarian p-JNK / JNK protein expression in each group; Figure 11J Changes in ovarian Cleaved Caspase-8 / Caspase-8 protein expression in each group; Figure 11K Changes in ovarian Cleaved Caspase-9 / Procaspase-9 protein expression in each group; Figure 11L Changes in ovarian Cleaved Caspase-3 / Caspase-3 protein expression in each group. **: P < 0.01 compared with the Con group; #: P < 0.05 compared with the TG group; ##: P < 0.01 compared with the TG group.

[0023] Figure 12 shows the effect of AE-n1 on spleen Th17 and Treg cells in rats with POI induced by TG ( n=8), Note: Figure 12A . Flow cytometry (FCM) analysis of the proportion of Th17 and Treg cells in the spleen cells of female rats in each group; Figure 12B .CD4 + IL-17A + Statistical graph of Th17 cell proportion; Figure 12C .CD4 + Foxp3 + Treg cell proportion statistics; Figure 12D .CD4 + IL-17A + Th17 / CD4 + Foxp3 + Statistical graph of Treg cell ratio. **: P < 0.01 compared with the Con group; ##: P < 0.01 compared with the TG group.

[0024] Figure 13 This is a graph showing the changes in body weight of SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0025] Figure 14 This is a graph showing changes in urine protein / creatinine ratios in SD rats with adriamycin-induced nephrotic syndrome in each group of the animal experiment of the present invention.

[0026] Figure 15 This is a graph of the kidney index of SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0027] Figure 16 This is a graph showing the liver index of SD rats with adriamycin-induced nephrotic syndrome in each group of the animal experiment of the present invention.

[0028] Figure 17 This is a graph showing the spleen index of SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0029] Figure 18 This is a graph of cardiac index intervention in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0030] Figure 19 The diagram is a pathological picture (200X) of the kidney tissues of SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment of the present invention.

[0031] Figure 20 This is a statistical graph of changes in serum urea nitrogen in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0032] Figure 21 This is a statistical graph of changes in serum creatinine in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0033] Figure 22 This is a statistical diagram of the changes in serum albumin in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0034] Figure 23 This is a statistical graph of changes in serum total cholesterol in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0035] Figure 24 This is a statistical diagram of changes in serum triglycerides in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0036] Figure 25 This is a western blot diagram of the changes in Cleave-caspase3 and pro-apoptotic protein Bax protein related to renal tissue apoptosis in SD rats with nephrotic syndrome induced by adriamycin in each group of the animal experiment part of the present invention.

[0037] Figure 26 This is a graph showing daily body weight changes of imiquimod-induced psoriasis BalB / c mice in each group in the animal experiment part of the present invention.

[0038] Figure 27This is a statistical graph of spleen index of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0039] Figure 28 These are pictures of skin lesions in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0040] Figure 29 This is a statistical graph showing the scores of scales at the skin lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0041] Figure 30 This is a statistical graph of the thickness of skin lesions in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0042] Figure 31 This is a statistical graph showing the erythema scores at the skin lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment of the present invention.

[0043] Figure 32 This is a statistical graph of the cumulative PASI scores of the skin lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0044] Figure 33 This is a statistical graph of the average skin thickness at the lesion site of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0045] Figure 34 This is a diagram showing the distribution of subcutaneous blood vessels in the lesions of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0046] Figure 35 These are diagrams showing the pathological changes in the skin lesions on the back of BalB / c mice with imiquimod-induced psoriasis in each group of the animal experiment of the present invention.

[0047] Figure 36 This is a graph showing the PCNA protein expression in the skin tissue of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0048] Figure 37 This is a graph showing changes in serum alanine aminotransferase (ALT) levels in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0049] Figure 38 This is a graph showing changes in serum aspartate aminotransferase (AST) levels in imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0050] Figure 39This is a graph showing changes in liver index of imiquimod-induced psoriasis BalB / c mice in each group in the animal experiment part of the present invention.

[0051] Figure 40 This is a graph showing changes in kidney index of imiquimod-induced psoriasis BalB / c mice in each group in the animal experiment part of the present invention.

[0052] Figure 41 This is a graph showing the changes in reproductive organ (ovary + fallopian tube + uterus) indexes of imiquimod-induced psoriasis BalB / c mice in each group of the animal experiment part of the present invention.

[0053] Figure 42 These are diagrams showing the morphological changes of the ovaries and uterus in each group of imiquimod-induced psoriasis BalB / c mice in the animal experiment part of the present invention. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below with reference to specific embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0055] Example 1 Preparation of Kunxining

[0056] The present invention extracts and separates Artemisia annua to prepare a high-content artemisinin component, and then adds artemisinin monomer to the high-content artemisinin component in a certain proportion to prepare an Artemisia annua extract rich in artemisinin and artemisinin, thereby obtaining Kunxining (abbreviated as AE-n1).

[0057] The Chinese medicinal material studied in this experiment is Artemisia annua, the dried aerial part of Artemisia annua, a plant of the Asteraceae family. It is native to Fengshun County, Meizhou City, Guangdong Province. It was harvested after flowering in 2018 and air-dried for later use.

[0058] Artemisinin, purity >98%, was purchased from Chongqing Kerui Pharmaceutical Co., Ltd.; Lot: P2160403; CAS: 63968-64-9.

[0059] Artemisitene (AT), purity 98%, CAS: 101020-89-7.

[0060] (1) Experimental methods

[0061] (1) Drug preparation: Extraction of Artemisia annua extract: Weigh 10 kg of dried Artemisia annua medicinal material, crush it, add 10 times the amount of petroleum ether (II) and soak it for 1 hour, heat it to 60°C, reflux it for 1.5 hours, and extract it 3 times in total; combine the extracts, and evaporate the solvent under reduced pressure to obtain 540 g of Artemisia annua extract, with an extract yield of 5.4%.

[0062] (2) Separation and preparation of high-content artemisinin components: 535g of Artemisia annua extract was mixed with 1 times the amount of 200-300 mesh silica gel, and 10 times the amount of 200-300 mesh silica gel was dry-packed on a column. The mixture was eluted with petroleum ether-ethyl acetate (100:0-80:20) and monitored by TLC. The artemisinin-containing fractions were collected, combined, and the solvent was recovered by vacuum rotary evaporation to obtain a mixture containing artemisinin. The artemisinin content was detected by ultra-high performance liquid chromatography and increased to about 30%. The obtained mixture component 84.8g was mixed with 2 times the amount of 200-300 mesh silica gel, and 20 times the amount of 200-300 mesh silica gel was dry-packed on a column. The mixture was eluted with petroleum ether-ethyl acetate (100:0-80:20) and monitored by TLC. The artemisinin-containing fractions were collected, combined, and the solvent was recovered by vacuum rotary evaporation to obtain 22.7g of the component mainly containing artemisinin. 5.0 g of the above-obtained fraction was mixed with twice the amount of 200-300 mesh silica gel and dry-packed onto a column using 20 times the amount of 200-300 mesh silica gel. Elution was performed with petroleum ether-ethyl acetate (100:0-80:20) and monitored by TLC. Fractions containing artemisinin were collected, combined, and the solvent was recovered by vacuum rotary evaporation to yield 1.9 g of a high-content artemisinin fraction. Ultra-high performance liquid chromatography (UPLC) analysis revealed an artemisinin content of approximately 85%.

[0063] (3) Determination of artemisinin content in high-content artemisinin fractions: 65 mg of high-content artemisinin fractions were weighed and dissolved in 1 mL of acetonitrile. After filtering through a 0.22 μm organic microporous filter membrane, the artemisinin content in the high-content artemisinin fractions was determined using UPLC analysis technology. Chromatographic conditions: Column: Waters Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm) (Waters, USA), mobile phase: A: water, B: acetonitrile; elution gradient: 0-15 min, 25% B; 15-25 min, 25%-55% B; 25-35 min, 55% B; 35-50 min, 55%-30% B; 50-53 min, 30% B; flow rate: 0.1 mL / min; elution time: 53 min; injection volume: 5.0 μL. Mass spectrometry detection conditions: Scan mode: Positive ion mode; Acquisition mode: Full scan (Full MS); Ion source: Electrospray ionization (HESI); Sheath gas volume flow rate: 40.0 abr; Auxiliary gas volume flow rate: 5.0 abr; Ion transfer capillary temperature: 320°C; Ion transfer capillary voltage: 6.0 V; Lens voltage: 90.0 V; Positive ion mode spray voltage: 5.0 kV; Mass resolution: 30,000; Mass scan range: m / z 100–1000. Data processing and analysis: Mass spectrometry data were processed and analyzed using Xcalibur 2.2 software. The retention time of artemisinin in the high-content artemisinin fraction was determined based on the retention time of the reference substance, and the artemisinin content was calculated based on the peak area and a binary regression equation.

[0064] (4) Adding an amount of artemisinin equivalent to artemisinin to the high-content artemisinin fraction: Weigh 33.05 mg of the high-content artemisinin fraction, calculate the artemisinin content of the high-content artemisinin fraction according to a linear equation, and then add an equal amount of artemisinin to the high-content artemisinin fraction to obtain an Artemisia annua extract rich in artemisinin and artemisinin, namely, Kunxining. Dissolve Kunxining in 1 mL of acetonitrile, filter, and analyze the content of each index component by HPLC.

[0065] (2) Experimental results

[0066] (1) HPLC method for detecting the content of artemisinin in high-content artemisinin fractions

[0067] 1. Prepare artemisinin acetonitrile standard solution: Dissolve 307 mg of artemisinin in 3 mL of acetonitrile to prepare an artemisinin acetonitrile standard solution with a concentration of 102.33 mg / mL. Dilute this solution again with chromatographic acetonitrile to concentrations of 40.93 mg / mL, 51.57 mg / mL, 61.40 mg / mL, and 81.87 mg / mL, respectively. After filtering the standard solutions through a 0.22 μm organic microporous filter, place them in a sample tray in ascending concentration order and inject them sequentially. Each sample is tested for 30 minutes.

[0068] 2. Draw the standard curve of artemisinin: According to the peak area of artemisinin standard solution, plot the concentration and fit the standard curve and linear equation. According to the corresponding relationship between the peak area and the concentration of artemisinin reference solution, fit the binary regression equation: y = 37.029x-1010, R 2 =0.9953. The fitting standard curve and linear equation obtained from the liquid phase data of pure artemisinin are as follows Figure 1 shown.

[0069] 3. Calculate the artemisinin content in the high-content artemisinin fraction: Prepare a 65 mg / mL acetonitrile solution of the high-content artemisinin fraction. The HPLC spectrum of the high-content artemisinin fraction is as follows: Figure 2 Substituting the artemisinin peak area of the high-content artemisinin fraction into the linear equation y=37.029x-1010, the artemisinin content was calculated to be 56.90 mg / mL, accounting for 87.54% of the high-content artemisinin fraction.

[0070] (2) Preparation of Kunxining and detection of index component content

[0071] 1. Preparation of Kunxining: Weigh 65.13 mg of a high-content artemisinin component, of which the artemisinin content is 65.13×87.54%=57.01 mg. An equal amount of artemisinin 57.01 mg needs to be added thereto. Therefore, 58.18 mg of artemisinin sample is added thereto (Note: the purity of the artemisinin sample is 98%) to obtain an Artemisia annua extract rich in artemisinin and artemisinin, namely, Kunxining.

[0072] 2. HPLC spectrum of Kunxining: The HPLC spectrum of Kunxining is as follows Figure 3 The peak with a retention time of 13.617 min represents artemisinin, the peak with a retention time of 14.974 min represents artemisinin, and the rest are impurity peaks. Due to the sensitivity of the ELSD detector, some impurity peaks are not displayed.

[0073] To explore the antagonistic effect and mechanism of Kunxining (AE-n1) on TG-induced ovarian insufficiency, an animal experiment on TG-induced premature ovarian insufficiency (POI) rats was conducted.

[0074] Experiment 1: Animal experiment on TG-induced premature ovarian insufficiency in rats

[0075] The experimental animals used in the experiment were SPF - level healthy female SD rats, purchased from the Guangdong Provincial Center for Laboratory Animals. The license numbers were: SCXK(Guangdong)2013 - 0002 and SCXK(Guangdong)2018 - 0002. The feeding environment of the experimental animals was as follows: maintained a 12 - hour cycle under light / dark conditions (lights on: 07:00 - 19:00), controlled the temperature (20 ± 2 °C), humidity (50 ± 10%), ventilation, with more than 10 air changes per hour (all fresh air systems), and allowed free access to standard rodent food and tap water. All animal experiment protocols were carried out in accordance with the "Regulations on the Administration of Laboratory Animals" promulgated by the National Science and Technology Commission and approved by the Laboratory Animal Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine; the ethical number of the experimental animals was: 2017057.

[0076] Experimental drug: AE - n1 prepared in Example 1. The mass ratio of artemisylene to artemisinin in AE - n1 was 1:1, and the mass ratio of artemisylene and artemisinin in AE - n1 was approximately 90%.

[0077] Other experimental reagents and instruments were all commercially available.

[0078] SPSS 19.0 was used for data statistical analysis. Measurement data were first subjected to a normality test. The data results that met the normal distribution were expressed as mean ± standard deviation Otherwise, the median and quartiles (M(P 25 ~P 75 )) were used for statistical description. For multiple groups of independent measurement data that met the normal distribution, one - way analysis of variance (one - way ANOVA) was used to compare the means. When one - way analysis of variance was statistically significant, pairwise comparisons of the means between groups were further carried out. When the variances were homogeneous, the least significant difference t - test (LSD - t test) was used to compare and analyze the differences in means between groups; when the variances were heterogeneous, Dunnett’s T3 test was used for statistical analysis. When the data did not meet the normal distribution, the Kruskal - Wallis non - parametric test was applied. Taking P < 0.05 as the standard for statistical significance. GraphPad Prism 6, Microsoft Office 2016, and Photoshop CC 2017 software were used for chart making.

[0079] The following is the specific experimental process:

[0080] (I) Drug preparation

[0081] (1) Preparation of AE-n1 solution: The daily dosage of Artemisia annua in clinical practice is 6-12 g. Here, we take the average of 9 g. The rat dosage is: 9 g / 60 kg × 6.25 (conversion coefficient) × 0.0863% (AE-n1 yield) = 0.8 mg / kg / d. The low dose (1 times the equivalent dose) = 0.8 mg / kg / d, the medium dose (2 times the equivalent dose) = 1.6 mg / kg / d, and the high dose (4 times the equivalent dose) = 3.2 mg / kg / d. Dissolve an appropriate amount of AE-n1 in 0.9% saline to prepare AE-n1 solutions with final concentrations of 0.16 mg / mL, 0.32 mg / mL, and 0.64 mg / mL, respectively.

[0082] (2) Preparation of TG solution: The dosage for rats was 75 mg / kg / d. An appropriate amount of TG was dissolved in 0.9% saline to prepare a TG solution with a final concentration of 15 mg / mL.

[0083] (3) Preparation of estradiol valerate (EV) solution: The rat dose is 0.1 mg / kg / day. Dissolve an appropriate amount of EV in 0.9% saline to prepare an EV solution with a final concentration of 0.02 mg / mL. Calculation of the EV rat dose: 1 mg / 60 kg (daily adult dose) × 6.25 (conversion factor) = 0.1 mg / kg / day.

[0084] (2) Grouping, modeling and drug administration methods

[0085] SPF healthy female SD rats were randomly divided into 6 groups after 3 days of adaptive feeding, with 8 rats in each group. The drug administration conditions of each group were as follows:

[0086] (1) Blank group (Con group): The mice were given an equal amount of 0.9% saline by gavage.

[0087] (2) Tripterygium wilfordii glycosides group (TG group): 75 mg / kg TG was administered by gavage.

[0088] (3) AE-n1 low-dose group (AE-n1-L group): 75 mg / kg TG and 0.8 mg / kg AE-n1 were administered by gavage.

[0089] (4) AE-n1 medium-dose group (AE-n1-M group): 75 mg / kg TG and 1.6 mg / kg AE-n1 were administered by gavage.

[0090] (5) AE-n1 high-dose group (AE-n1-H group): 75 mg / kg TG and 3.2 mg / kg AE-n1 were administered by gavage.

[0091] (6) Positive drug group (EV group): 75 mg / kg TG and 0.1 mg / kg estradiol valerate (hereinafter referred to as EV) were administered by gavage.

[0092] Between 9:00 and 11:00 AM daily, the TG, AE-n1-L, AE-n1-M, AE-n1-H, and EV groups were gavaged with 75 mg / kg TG to establish the model. 0.5 hour after model establishment, the AE-n1-L, AE-n1-M, and AE-n1-H groups were gavaged with AE-n1 at 0.8 mg / kg, 1.6 mg / kg, and 3.2 mg / kg, respectively. The EV group was gavaged with 0.1 mg / kg EV. The Con group was gavaged with an equal volume of 0.9% saline. Model establishment and drug administration were continued once daily for 28 days. No food or water were allowed for 24 hours before dissection.

[0093] (3) Observation indicators

[0094] (1) General information: including vaginal exfoliated cell smear and HE staining, sampling, calculation of ovarian index and uterine index, detection of serum sex hormone indicators (LH, FSH, E2, AMH), embedding and HE staining, TUNEL staining, and transmission electron microscopy, as shown in ① to ⑧:

[0095] ① Vaginal exfoliated cell smear and HE staining

[0096] During the drug intervention period, vaginal exfoliated cell smears were performed at 8:00-9:00 am every day. The specific operation was as follows: 12 μL of 0.9% normal saline was drawn up with a pipette, placed in the mouse vagina, and repeatedly aspirated three times, and then evenly smeared on a glass slide.

[0097] After the smear is dry, HE staining is performed. The specific steps are as follows:

[0098] Rehydrate the slides (xylene I → xylene II → 100% ethanol → 95% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → 50% ethanol → water, soaking for 1 minute each). Stain with hematoxylin for 3 minutes, rinse with running water for 3 minutes. Separate the slides with color separation solution for a few seconds. Dehydrate (50% ethanol → 70% ethanol → 80% ethanol → 90% ethanol, soaking for 1 minute each). Stain with eosin for 30 seconds. Dehydrate (95% ethanol → 100% ethanol → xylene II → xylene I, soaking for 2 minutes each). Mount the slides with neutral gum.

[0099] Based on the results of vaginal exfoliated cell images, the estrous cycle changes of mice were observed. The following criteria for the estrous cycle changes of normal rats were used for judgment according to the "Medical Laboratory Animal Science":

[0100] The mouse estrous cycle typically lasts 4-5 days. During proestrus, images show numerous nucleated epithelial cells and a small number of cornified epithelial cells, lasting 9-18 hours. During estrus, smears show numerous cornified epithelial cells in a "falling leaf" pattern, with few nucleated epithelial cells. This lasts 6-12 hours. During metestrus, smears show cornified epithelial cells, nucleated epithelial cells, and leukocytes, lasting 30-48 hours. During diestrus, smears show numerous leukocytes and few nucleated epithelial cells, lasting 36-42 hours.

[0101] ②Acquisition of materials

[0102] The mice were dissected 24 hours after the last administration, and were fasting but not watering within 24 hours. During the dissection, the mice were weighed first, anesthetized by intraperitoneal injection of 45 mg / kg of sodium phenobarbital, and the eyeballs were removed to collect blood. The whole blood was temporarily stored in a coagulant blood collection tube, allowed to stand for more than 4 hours, centrifuged at 3000 rpm for 15 minutes, and the upper serum was taken and stored in a -80°C refrigerator. The bilateral ovaries and uterus were weighed, and the uterus and one ovary were fixed in 4% paraformaldehyde for 24 hours, dehydrated, embedded and sliced, and HE staining, immunohistochemical staining and TUNEL staining were performed. Three different parts of the other ovary were cut into 1 mm3 strips, fixed in glutaraldehyde, and examined by transmission electron microscopy.

[0103] ③Calculation of ovarian index and uterine index

[0104] Ovarian index = ovary weight (wet weight) / mouse body weight × 100%

[0105] Uterine index = uterine weight (wet weight) / mouse body weight × 100%

[0106] ④ Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH) were detected by enzyme-linked immunosorbent assay (ELISA).

[0107] ⑤Embedding and sectioning

[0108] Tissues were fixed in 4% paraformaldehyde for 24 hours and then dehydrated in an automatic dehydrator. Immediately after dehydration, the tissues were immersed in the wax chamber of an embedding table for 10 minutes before embedding. Tissue blocks were sectioned into 3 μm thick sections, spread in a 42°C water bath, attached to adhesive slides, and air-dried at room temperature before staining.

[0109] ⑥HE staining

[0110] Oven-dry the slides at 60°C for 1 hour. Dewax (xylene I → xylene II, immersion for 10 minutes each). Rehydrate (100% ethanol → 100% ethanol → 95% ethanol → 95% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → 50% ethanol → 20% ethanol → water, immersion for 5 minutes each). Stain with hematoxylin for 10 minutes, rinse with running water for 5 minutes. Separate the slides with color separation solution for a few seconds. Dehydrate: 20% ethanol → 50% ethanol → 70% ethanol → 80% ethanol → 90% ethanol, immersion for 2 minutes each. Stain with eosin for 3 minutes. Dehydrate (95% ethanol → 100% ethanol → xylene II → xylene I, 5 minutes each). Mount the slides with neutral gum.

[0111] ⑦TUNEL fluorescence staining

[0112] Bake the slides in a 60°C oven for 1 hour. Dewax (xylene I → xylene II, 10-min soak each). Rehydrate (100% ethanol → 100% ethanol → 95% ethanol → 95% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → 50% ethanol → 20% ethanol → water, 5-min soak each). Circle the slides for immunohistochemistry. Add proteinase K (20 μg / mL), incubate at 37°C for 30 minutes, and wash with PBS for 5 minutes × 5 times. Add TUNEL staining reagent, incubate at 37°C for 1 hour, and wash with PBS for 5 minutes × 5 times. Stain with DAPI, incubate at room temperature in the dark for 5 minutes, and wash with PBS for 5 minutes × 5 times. Add anti-fluorescence quenching mounting solution, seal the slides, and observe under a fluorescence microscope.

[0113] ⑧ Transmission electron microscopy inspection: The transmission electron microscope is sent to an external company with inspection qualifications for inspection.

[0114] (2) Western Blot detection:

[0115] ① Tissue protein extraction: Thaw ovarian tissue on ice, add 500 μL of tissue protein extraction reagent containing protease and phosphatase inhibitors, and homogenize. After homogenization, centrifuge at 12,000 rpm at 4°C for 15 minutes, and collect the supernatant. After measuring protein concentration using the BCA assay, adjust the protein concentration of all samples to a consistent volume of 160 μL. Add 40 μL of 5× Loading Buffer, denature at 100°C for 10 minutes, and store in a refrigerator at -80°C until needed. Select a suitable brand of 10% or 12% premixed polyacrylamide gel preparation solution and prepare the gel for the experiment according to the instructions. While waiting for the gel to solidify, prepare the SDS-PAGE gel electrophoresis buffer in advance. Place the prepared gel in the electrophoresis tank and add the electrophoresis buffer. Add 10-30 μL of sample to each well of the sample well. Run the sample through the stacking gel at 80 V. Then adjust the voltage to 120 V to run the sample through the entire gel to complete the SDS-PAGE gel electrophoresis.

[0116] ② Transfer operation (PVDF membrane, pre-wetted in methanol for 5 minutes in advance), transfer conditions are: 300mA, 120-150min (corresponding to the time selection of the dividing line of molecular weight of 100KDa, among which the selected transfer time for the separated proteins with a molecular weight of less than 100KDa is 120min, and the selected transfer time for proteins with a molecular weight greater than 100KDa is 150min); after the transfer is completed, block with 5% skim milk or 6% BSA for 2h and incubate the primary antibody according to the corresponding strip in a refrigerator at 4℃ overnight; the next day, wash away the unbound primary antibody with TBST washing solution (wash 3 times, 5min each time); incubate with secondary antibody at 37℃ for 2h, and wash away the unbound secondary antibody with TBST washing solution (wash 3 times, 5min each time); expose the target strip with ECL luminescent liquid as a substrate, analyze, record and statistically analyze the results.

[0117] (3) Detection of the number of Th17 and Treg cells in spleen tissue by flow cytometry

[0118] Preparation of spleen single cell suspension: After sampling, immediately place the spleen tissue in pre-cooled RPMI-1640 culture medium and transfer to a clean bench. After the fat tissue around the spleen is peeled off, place it in a 200-mesh filter, add 5 mL of RPMI-1640 culture medium, and grind the spleen tissue with a 2 mL syringe needle to release spleen cells. Collect the spleen single cell suspension, centrifuge at 300g for 5 minutes, and discard the supernatant. Add 3 mL of red blood cell lysis buffer, mix gently, lyse at room temperature in the dark for 10 minutes, centrifuge at 400g for 5 minutes, and lyse twice. Add 1 mL of Stain Buffer and wash once, centrifuge at 400g for 5 minutes. Resuspend with RPMI-1640 culture medium containing 10% FBS, count the cells, and adjust the cell concentration so that the cell concentration is not less than 5×10 6 cells / mL.

[0119] Flow cytometry was used to detect the number of Th17 and Treg cells in spleen cells: the prepared spleen cell suspension was taken, the stimulation mixture was added to the spleen cell suspension at a ratio of 2 L / mL, the suspension was gently pipetted to mix, and the cells were seeded into a 96-well plate to ensure that the number of cells in each well was not less than 1 × 10 6Stimulate cells in a 37°C, 5% CO2 incubator for 10 hours. Transfer cells to flow cytometry tubes, resuspend in 1 mL of Stain Buffer, centrifuge at 400 g for 5 minutes, and discard the supernatant. Resuspend cells in 100 μL of PBS. Add 0.5 μL of CD4-FITC and 0.5 μL of CD25-APC surface antibodies, and incubate at 4°C in the dark for 30 minutes. Wash once with 1 mL of Stain Buffer, centrifuge at 400 g for 5 minutes, and discard the supernatant. Permeabilize the membrane with 1 mL of Fix / Perm Buffer, and incubate at 4°C in the dark for 30 minutes. Wash once with 1 mL of Perm Buffer, centrifuge at 400 g for 5 minutes, and discard the supernatant. Resuspend cells in 100 μL of PBS. Add 5 μL of Foxp3-PE and 0.5 μL of IL-17A-APC intracellular antibodies, and incubate at 4°C in the dark for 30 minutes. Wash once with 1 mL of Stain Buffer, centrifuge at 400 g for 5 minutes, and discard the supernatant. Add 300 μL PBS to resuspend and detect on the instrument.

[0120] (IV) Experimental results

[0121] The following are the results and analysis of the effects of AE-n1 on the ovary and uterus, estrous cycle, serum sex hormone levels, ovarian tissue morphology, ovarian follicles at all levels, ovarian ultrastructure, ovarian granulosa cell apoptosis, ovarian apoptosis-related protein expression, and spleen Th17 and Treg cells in rats with POI caused by TG.

[0122] (1) Effects on the ovaries and uterus of rats with POI induced by TG

[0123] Figure 4 The diagrams of changes in the ovaries and uterus of rats in each group are shown below. Figure 4 A. Anatomical diagram of the ovary-fallopian tube-uterus of rats in each group, Figure 4 B. Ovarian index of rats in each group, Figure 4 C. Uterine index of rats in each group. **: P < 0.01 compared with the Con group; ##: P < 0.01 compared with the TG group.

[0124] Depend on Figure 4 As shown in A, compared with the Con group, the ovaries and uterus of the TG group rats were significantly thickened and enlarged, the oviducts were thickened or elongated, the tube walls were thinned, and some rats had hydrops in the oviducts; while the damage to the ovaries, uterus and oviducts of the female rats in the AE-n1-L, AE-n1-M, AE-n1-H and EV groups was significantly improved. Figure 4 B. Figure 4As shown in Figure C, compared with the Con group, the ovarian index and uterine index of the rats in the TG group were significantly increased, with significant differences (P < 0.01). The AE-n1-L, AE-n1-M, and AE-n1-H groups could ameliorate the TG-induced increase in the ovarian index of rats (P < 0.01) and significantly ameliorate the TG-induced increase in the uterine index of rats (P < 0.01). Compared with the TG group, the ovarian index of the rats in the EV group decreased (P < 0.01), and the uterine index decreased significantly (P < 0.01).

[0125] (2) Effects on the estrous cycle of TG-induced POI rats

[0126] Figure 5 The graph shows the effect of AE-n1 on the estrous cycle of TG-induced POI rats (M (P25-P75), n=8). Note: **: P < 0.01 compared with the Con group; #: P < 0.05 compared with the TG group; ##: P < 0.01 compared with the TG group.

[0127] Depend on Figure 5 As can be seen, compared with the Con group, the number of estrous cycles in the TG group was significantly reduced, with a significant difference (P < 0.01). Compared with the TG group, the number of abnormal estrous cycles in rats in all AE-n1 dose groups was significantly reduced, with statistically significant differences (P < 0.05 or P < 0.01), and the difference was dose-dependent. Furthermore, the number of abnormal estrous cycles in rats in the EV group was also significantly reduced, with a significant difference compared with the TG group (P < 0.01).

[0128] (3) Effects on serum sex hormone levels in rats with POI induced by TG

[0129] Figure 6 The effect of AE-n1 on serum sex hormone levels in TG-induced POI rats ( n=8), Note: Figure 6 A. Serum LH levels of rats in each group; Figure 6 B. Serum FSH levels of rats in each group; Figure 6 C. Serum E2 levels of rats in each group; Figure 6 D. Serum AMH levels of rats in each group. **: P < 0.01 compared with the Con group; #: P < 0.05 compared with the TG group; ##: P < 0.01 compared with the TG group.

[0130] Depend on Figure 6As shown in Figure A, compared with the Con group, the serum LH levels of rats in the TG group were significantly increased (P < 0.01). Compared with the TG group, the serum LH levels of rats in the medium- and high-dose AE-n1 groups were significantly decreased (P < 0.01), and the serum LH levels of rats in the AE-n1-L group were significantly decreased (P < 0.05). In addition, the serum LH levels of rats in the EV group were also significantly decreased, with a significant difference compared with the TG group (P < 0.01).

[0131] Depend on Figure 6 As shown in Figure B, TG significantly induced an increase in serum FSH levels in rats, with a significant difference compared to the Con group (P < 0.01). Compared to the TG group, all doses of AE-n1 reversed the increase in serum FSH levels in rats, with significant differences (P < 0.01) in a dose-dependent manner. Serum FSH levels in rats in the EV group were also significantly decreased, with a significant difference compared to the TG group (P < 0.01).

[0132] Depend on Figure 6 As shown in Figure 3, compared with the Con group, TG significantly reduced the serum E2 level in rats (P < 0.01). Compared with the TG group, the AE-n1-M and AE-n1-H groups significantly improved the inhibitory effect of TG on the serum E2 level in rats (P < 0.01), while the AE-n1-L group had no significant inhibitory effect on the TG-induced decrease in serum E2 levels in rats (P > 0.05). EV also significantly reversed the decrease in serum E2 levels in rats (P < 0.01).

[0133] Depend on Figure 6 As shown in Figure 4, TG significantly reduced serum AMH levels in rats, with a significant difference compared to the Con group (P < 0.01). The serum AMH levels in rats in the medium- and high-dose AE-n1 groups were significantly increased compared to the TG group (P < 0.01) in a dose-dependent manner. There was no significant difference in serum AMH levels between the AE-n1-L group and the TG group (P > 0.05). EV also significantly antagonized the TG-induced decrease in serum AMH levels in rats, with a significant difference compared to the TG group (P < 0.01).

[0134] (4) Effects on ovarian tissue morphology in rats with POI induced by TG

[0135] Figure 7 The effect of AE-n1 on the ovarian tissue morphology of TG-induced POI rats (200×). Note: Figure 7 A. HE staining results of ovarian tissue of rats in Con group; Figure 7 B. HE staining results of ovarian tissue of rats in TG group; Figure 7C. HE staining results of ovarian tissue of rats in AE-n1-L group; Figure 7 D. HE staining results of ovarian tissue of rats in AE-n1-M group; Figure 7 E. HE staining results of ovarian tissue of rats in AE-n1-H group; Figure 7 F. HE staining results of ovarian tissue of rats in EV group.

[0136] Depend on Figure 7 As can be seen, all follicles and corpora lutea at all levels were visible in the ovaries of female rats in the Con group, indicating well-developed ovaries, normal cortical morphology, and intact tissue structure. In contrast, the ovaries of female rats in the TG group contained fewer primary, secondary, and mature follicles, and relatively more atretic follicles. Primordial follicles and corpora lutea were no different from those in the Con group. The ovarian cortex of rats in the low-, medium-, and high-dose AE-n1 groups, as well as the EV group, was acceptable, with good morphology and intact ovarian tissue. A certain number of primordial follicles, primary, secondary, mature follicles, and corpora lutea were visible in the cortex, while atretic follicles were relatively rare.

[0137] (5) Effects on ovarian follicles at all levels in rats with POI induced by TG

[0138] Figure 8 The diagram shows the effect of AE-n1 on ovarian follicles at all levels in TG-induced POI rats (M (P25-P75), n=8). Note: *: P < 0.05 compared with the Con group; **: P < 0.01 compared with the Con group; #: P < 0.05 compared with the TG group; ##: P < 0.01 compared with the TG group.

[0139] Depend on Figure 8 visible:

[0140] ① Comparison of primordial follicle numbers: There was no significant difference in the number of primordial follicles among the groups (P>0.05), indicating that the intervention of TG, AE-n1 and EV had no effect on the number of primordial follicles in the ovaries of SD female rats.

[0141] ② Comparison of primary follicle numbers: TG led to a significant decrease in the number of primary follicles in the ovaries of rats, with a significant difference compared to the Con group (P < 0.01). High-dose AE-n1 intervention reversed the decrease in the number of primary follicles, with a significant difference compared to the TG group (P < 0.05). Compared to the TG group, the EV group also significantly increased the number of primary follicles in the ovaries of rats, with a statistically significant difference (P < 0.05).

[0142] ③ Comparison of the number of secondary follicles: Compared with the Con group, the number of secondary follicles in the ovaries of rats in the TG group was significantly reduced, and the difference was significant (P < 0.01); the number of secondary follicles in the ovaries of rats in the medium and high doses of AE-n1 groups and the EV group was significantly increased compared with the TG group (P < 0.05), indicating that the intervention of medium and high doses of AE-n1 and EV can improve the trend of TG-induced decrease in the number of secondary follicles in rats.

[0143] ④ Comparison of the number of mature follicles: The number of mature follicles in the ovaries of rats in the TG group was significantly reduced compared with that in the Con group, and the difference was statistically significant (P < 0.05); medium and high doses of AE-n1 could reverse the decrease in the number of mature follicles in the ovaries of rats caused by TG (P < 0.05), and EV could also significantly improve the trend of decrease in the number of mature follicles in the ovaries of rats induced by TG (P < 0.05).

[0144] ⑤ Comparison of corpus luteum number: There was no significant difference in the number of corpus luteum in the ovaries of rats in the TG group, AE-n1 high-, medium-, and low-dose groups, and EV group (P>0.05), indicating that the intervention of TG, AE-n1, and EV had no effect on the original number of corpus luteum in the ovaries of SD female rats.

[0145] ⑥ Comparison of the number of atretic follicles: Compared with the Con group, TG significantly increased the number of atretic follicles in the rat ovaries (P < 0.01). Compared with the TG group, the number of atretic follicles in the low- and medium-dose AE-n1 groups was significantly reduced (P < 0.05), and the number of atretic follicles in the high-dose AE-n1 group was significantly reduced (P < 0.01). In addition, the EV group also significantly reversed the trend of increased atretic follicles in the ovaries of rats caused by TG, with a significant difference compared with the TG group (P < 0.05).

[0146] These results suggest that AE-n1 can stimulate the development and maturation of ovarian follicles in rats with TG-induced ovarian dysfunction, promoting an increase in the number of primary, secondary, and mature follicles and improving ovarian function. Furthermore, EV can also improve TG-induced ovarian dysfunction in rats and maintain normal ovarian function.

[0147] (6) Effects on the ultrastructure of the ovary in rats with POI induced by TG

[0148] Figure 9 The effect of AE-n1 on the ultrastructure of ovary in rats with POI induced by TG (5000×-30000×).

[0149] Depend on Figure 9As can be seen, follicles were visible in the ovarian cortex of rats in the Con group, and multiple layers of granulosa cells were visible in the granulosa layer. The cells were neatly arranged, with intercellular spaces visible. The cells were predominantly euchromatin, and the cells were plump. The cytoplasm contained abundant mitochondria, free ribosomes, and rough endoplasmic reticulum. The nuclei were elongated, spindle-shaped, or oval, with distinct nucleoli. The ovarian ultrastructure of rats in the TG group was damaged to some extent, with thinning of the granulosa cell layer, local cell loss, lipid droplet formation in the cytoplasm, swollen mitochondria, irregular granulosa cells, condensed and marginalized chromatin in some areas, and dilated rough endoplasmic reticulum. In the ovarian cortex of rats in the low- and medium-dose AE-n1 groups, the cells were arranged in an orderly manner, with slightly irregular nuclei, abundant cytoplasm, slightly dilated rough endoplasmic reticulum, slightly dilated mitochondrial cristae, and a small amount of condensed chromatin. The ovarian cortical cells of rats in the AE-n1-H and EV groups were arranged in a slightly orderly manner, with a relatively abundant granulosa cell layer. The mitochondria in the cytoplasm were slightly swollen, and the rough endoplasmic reticulum did not show obvious expansion. The nuclei were long spindle-shaped or oval, mainly composed of euchromatin, and gaps were visible between some cells.

[0150] These results suggest that AE-n1 can alleviate TG-induced changes in rat ovarian ultrastructure to a certain extent, antagonize TG ovarian toxicity, protect the internal structure of follicles and oocytes, and thus improve ovarian function. Furthermore, EV can also play a protective role against TG-induced changes in ovarian ultrastructure to a certain extent.

[0151] (7) Effects on apoptosis of ovarian granulosa cells in rats with POI induced by TG

[0152] Figure 10 Effect of AE-n1 on apoptosis of ovarian granulosa cells in rats with POI induced by TG (200×)( n=8), Note: Figure 10A .TUNEL expression in granulosa cells of ovarian follicles in rats of each group (200×); Figure 10B Statistical graph of the mean fluorescence intensity of TUNEL in granulosa cells in the ovarian tissue of rats in each group. **: P < 0.01 compared with the Con group; ##: P < 0.01 compared with the TG group.

[0153] Depend on Figure 10AIt can be seen that the cell nuclei in the rat ovary were successfully stained and can be used for positive staining positioning; the positive areas of TUNEL fluorescence staining were mostly found in the granulosa cells in the follicles in the rat ovarian tissue, and the positive staining parts in the ovaries of rats in each group were mostly located in the same area. The positive parts of TUNEL fluorescence staining were mostly stained on the nuclei of granulosa cells, while the extranuclear areas were rarely stained, indicating that the detection rate of TUNEL fluorescence staining positive areas in rat ovaries was high, the non-specific binding signals were small, and the results were reliable. Compared with the Con group, the TUNEL binding fluorescence signal in the TG group increased significantly, and the TUNEL immunofluorescence binding signals in the high, medium, and low dose groups of AE-n1 and the EV group decreased to varying degrees. The specific analysis results are as follows:

[0154] Depend on Figure 10B Compared with the Con group, the TUNEL fluorescence signal in ovarian granulosa cells of rats in the TG group increased significantly, with a statistically significant difference (P < 0.01), indicating that TG can significantly induce apoptosis of rat ovarian granulosa cells. Compared with the TG group, the TUNEL fluorescence signal in ovarian granulosa cells of rats in the high-, medium-, and low-dose AE-n1 groups decreased significantly, with significant differences (P < 0.01), and was dose-dependent, indicating that AE-n1 can antagonize TG-induced apoptosis of rat ovarian granulosa cells, and this anti-apoptotic effect increased with increasing drug dose. The TUNEL fluorescence signal in ovarian granulosa cells of rats in the EV group also decreased significantly, with a significant difference compared with the TG group (P < 0.01).

[0155] (8) Effects on the expression of ovarian apoptosis-related proteins in TG-induced POI rats

[0156] Figure 11 The effect of AE-n1 on the expression of apoptosis-related proteins in ovarian apoptosis rats induced by TG (x±s, n=8). Note: Figure 11A Changes in TNF-R1 protein expression in the ovaries of each group; Figure 11B .Changes in ovarian Cyto-C protein expression in each group; Figure 11C .Changes in Apaf-1 protein expression in the ovaries of each group; Figure 11D Changes in XIAP protein expression in the ovaries of each group; Figure 11E .Changes in the expression of Bax and Bcl-2 proteins in the ovaries of each group; Figure 11F .Changes in ovarian Bax protein expression in each group; Figure 11G .Changes in Bcl-2 protein expression in the ovaries of each group; Figure 11H .Changes in the Bax / Bcl-2 ratio in the ovaries of each group; Figure 11I Changes in ovarian p-JNK / JNK protein expression in each group; Figure 11J Changes in ovarian Cleaved Caspase-8 / Caspase-8 protein expression in each group; Figure 11KChanges in ovarian Cleaved Caspase-9 / Procaspase-9 protein expression in each group; Figure 11L Changes in ovarian Cleaved Caspase-3 / Caspase-3 protein expression in each group. **: P < 0.01 compared with the Con group; #: P < 0.05 compared with the TG group; ##: P < 0.01 compared with the TG group.

[0157] Depend on Figure 11 Results: TG increased the expression of Cyto-C and APaf-1 proteins and the Cleaved Caspase-9 / Procaspase-9 ratio in the rat ovaries, with statistically significant differences compared to the Con group. AE-n1 antagonized the effects of TG on the ovaries, decreasing the expression of Cyto-C and Apaf-1 proteins and the Cleaved Caspase-9 / Procaspase-9 ratio. Compared to the Con group, XIAP protein expression in the ovaries of the TG group was significantly decreased. High-dose AE-n1 administration significantly increased XIAP protein expression compared to the TG group. Compared to the Con group, TG induced increased Cleaved Caspase-3 protein expression in the rat ovaries, leading to increased ovarian cell apoptosis. AE-n1 reduced Cleaved Caspase-3 protein expression and significantly decreased the apoptosis rate of ovarian granulosa cells.

[0158] (9) Effects on Th17 and Treg cells in the spleen of rats with POI induced by TG

[0159] Figure 12 shows the effect of AE-n1 on spleen Th17 and Treg cells in rats with POI induced by TG (x±s, n=8). Note: Figure 12A . Flow cytometry (FCM) analysis of the proportion of Th17 and Treg cells in the spleen cells of female rats in each group;

[0160] Figure 12B .CD4 + IL-17A + Statistical graph of Th17 cell proportion; Figure 12C .CD4 + Foxp3 + Treg cell proportion statistics; Figure 12D .CD4 + IL-17A + Th17 / CD4 + Foxp3 +Statistical graph of Treg cell ratio. **: P < 0.01 compared with the Con group; ##: P < 0.01 compared with the TG group.

[0161] As shown in Figure 12, in the rat ovarian insufficiency model, TG can significantly induce CD4 + IL-17A + Increased proportion of Th17 cells and CD4 + Foxp3 + The decrease in the proportion of Treg cells led to an imbalance in the Th17 / Treg ratio, and the difference was statistically significant compared with the Con group (P < 0.01). + IL-17A + Th17 cell ratio and increased CD4 + Foxp3 + The proportion of Treg cells was significantly decreased, but there was no statistical difference (P>0.05). + Foxp3 + The proportion of Treg cells was significantly decreased, thereby downregulating the Th17 / Treg ratio. The difference was statistically significant compared with the TG group (P < 0.01). High-dose AE-n1 could significantly reduce the CD4 + IL-17A + Th17 cell ratio, increased CD4 + Foxp3 + The proportion of Treg cells was significantly decreased in the EV group, thereby downregulating the Th17 / Treg ratio. The difference was statistically significant compared with the TG group (P < 0.01). Similarly, EV can also reverse the CD4 + IL-17A + Increased proportion of Th17 cells, CD4 + Foxp3 + The proportion of Treg cells decreased and the Th17 / Treg ratio increased.

[0162] The above results indicate that in the TG-induced ovarian insufficiency rat model, spleen CD4 + IL-17A + Increased proportion of Th17 cells, CD4 + Foxp3 + The decrease in the proportion of Treg cells and the increase in the Th17 / Treg ratio caused the imbalance of immune homeostasis in rats. The intervention of AE-n1 can reduce the CD4 + IL-17A + Th17 cell ratio, increased CD4 + Foxp3 +The proportion of Treg cells and the reduction of Th17 / Treg ratio can reverse the pathological state of immune regulation disorder in rats, thereby playing a certain therapeutic role in the disease state of ovarian insufficiency induced by TG in rats.

[0163] In summary, AE-n1 can improve the toxic state of premature ovarian insufficiency caused by triglycerides in SD rats, reduce ovarian granulosa cell apoptosis, improve ovarian pathology, promote follicular development, restore serum sex hormone levels to normal to a certain extent, improve the imbalance of Th17 / Treg immune cells, and restore the estrous cycle of rats to a certain extent. The mechanism of AE-n1's antagonistic effect on triglycerides-induced ovarian insufficiency in rats is as follows:

[0164] (1) The present invention reflects ovarian function by detecting the levels of rat serum sex hormones LH, FSH, E2, and AMH. The results show that AE-n1 can effectively reduce serum LH and FSH levels and increase serum E2 and AMH levels, thereby improving the POI disease state of SD rats caused by Trichoderma lucidum. This suggests that AE-n1 exerts its ovarian protective effect by regulating hormone levels.

[0165] (2) The present invention detected the expression of apoptosis-related proteins in rat ovaries. The results showed that TG induced an increase in the expression of TNF-R1, Caspase-8, JNK, Bax, Cyto-C, and Apaf-1 proteins in rat ovaries, and inhibited the expression of Bcl-2 and XIAP proteins. AE-n1 can alleviate the symptoms of POI induced by TG by reducing the expression of TNF-R1, Caspase-8, JNK, Bax, Cyto-C, and Apaf-1 proteins and increasing the expression of Bcl-2 and XIAP proteins. This suggests that AE-n1 alleviates the apoptosis of ovarian granulosa cells caused by TG through the TNF-R1-mediated apoptosis signaling pathway, thereby reducing the toxicity of TG to the ovary.

[0166] (3) The present invention also analyzed the changes in the ratio of Th17 and Treg cells in the spleen of rats. The results showed that TG could induce an increase in the ratio of Th17 cells and a decrease in the ratio of Treg cells in the spleen of rats, causing a serious imbalance in the Th17 / Treg ratio. This indicates that the imbalance in the Th17 / Treg ratio in the spleen may be involved in the occurrence and development of ovarian insufficiency in rats. The combined administration of AE-n1 and TG can reduce the ratio of Th17 cells in the spleen of rats and increase the ratio of Treg cells, so that the Th17 / Treg ratio is effectively restored. This suggests that AE-n1 antagonizes the toxicity of TG by restoring the imbalance of Th17 / Treg cells in the spleen, thereby alleviating the immune disorder induced by TG in rats and improving the toxic state of ovarian insufficiency in rats.

[0167] In addition, to explore the effect of Kunxining on the physiological activity of tripterygium glycosides, the present invention compounded Kunxining with tripterygium glycosides to obtain a tripterygium glycosides composition, and then carried out a pharmacodynamic animal experiment on the treatment of adriamycin-induced nephrotic syndrome with the tripterygium glycosides composition and a pharmacodynamic animal experiment on the treatment of psoriasis with the tripterygium glycosides composition.

[0168] Experiment 2: Pharmacodynamic animal experiment on adriamycin-induced nephrotic syndrome

[0169] I. Experimental animals

[0170] The experimental animals used in the experiment were SPF-grade healthy male SD rats, purchased from the Guangdong Provincial Center for Medical Experimental Animals, with the license numbers: SCXK(Guangdong)2013-0002, SCXK(Guangdong)2018-0002. The feeding environment of the experimental animals was as follows: maintained in a 12-hour cycle under light / dark conditions (light on: 07:00-19:00), controlled temperature (20±2°C), humidity (50±10%), ventilation, with more than 10 air changes per hour (all fresh air systems), and allowed free access to standard rat food and tap water. All animal experiment protocols were carried out in accordance with the "Regulations on the Administration of Experimental Animals" promulgated by the State Science and Technology Commission and approved by the Experimental Animal Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine; the experimental animal ethics number: 2020004.

[0171] II. Drug preparation

[0172] Tripterygium glycosides was dissolved in pure water to prepare a 2mg / mL tripterygium glycosides solution for standby.

[0173] The Kunxining (AE-n1) prepared in Example 1 was added to pure water and ultrasonically suspended to obtain an Artemisia annua extract solution for standby.

[0174] Separate 2mg / mL tripterygium glycosides solution, and appropriate amounts of the above Artemisia annua extract solution were added to the tripterygium glycosides solution according to the concentration of Artemisia annua extract in the solution of 0.021mg / ml, 0.042mg / ml, and 0.085mg / ml respectively to obtain a low-dose tripterygium glycosides composition solution, a medium-dose tripterygium glycosides composition solution, and a high-dose tripterygium glycosides composition solution.

[0175] Prednisone: The dosing dose for rats was 6.3mg / kg / day, and it was configured into a 1.26mg / mL prednisone solution using pure water.

[0176] III. Grouping, model establishment and dosing method

[0177] SPF healthy male SD rats were randomly divided into 7 groups after 3 days of adaptive feeding: blank group (Control), model group (Model), tripterygium wilfordii polyglycosides group (TG), low-dose tripterygium wilfordii polyglycosides combination group (LXHK-L), medium-dose tripterygium wilfordii polyglycosides combination group (LXHK-M), high-dose tripterygium wilfordii polyglycosides combination group (LXHK-H) and prednisone group (Prednisone), with 6 rats in each group.

[0178] Except for the blank group, each group of rats received a single tail vein injection of doxorubicin 6 mg / kg. Three weeks later, the modeling group began to receive the drug according to the urine protein / creatinine ratio. The drug was given for 5 consecutive weeks. The dosage of each group was as follows:

[0179] Blank group (Control): The mice were given an equal amount of pure water by gavage every day according to their body weight;

[0180] Model group (Model): Administer an equal amount of pure water by gavage daily according to body weight;

[0181] Tripterygium wilfordii polyglycosides group (TG): 10 mg / kg / day of Tripterygium wilfordii polyglycosides solution was given orally;

[0182] The low-dose tripterygium wilfordii polyglycosides combination group (LXHK-L) was given a low-dose tripterygium wilfordii polyglycosides combination solution by oral gavage at a dose of 10 mg / kg / day of tripterygium wilfordii polyglycosides and 0.106 mg / kg / day of Artemisia annua extract.

[0183] The medium-dose tripterygium wilfordii polyglycosides combination group (LXHK-M) was given a medium-dose tripterygium wilfordii polyglycosides combination solution orally at a dose of 10 mg / kg / day of tripterygium wilfordii polyglycosides and 0.213 mg / kg / day of Artemisia annua extract;

[0184] The high-dose tripterygium wilfordii polyglycosides combination group (LXHK-H) was given a high-dose tripterygium wilfordii polyglycosides combination solution by oral gavage at a dose of 10 mg / kg / day of tripterygium wilfordii polyglycosides and 0.426 mg / kg / day of Artemisia annua extract.

[0185] Positive drug prednisone group (Prednisone): Prednisone solution was administered orally at a dose of 6.3 mg / kg / day.

[0186] 4. Statistical Methods

[0187] GraphPad Prism 8.0.2 was used for statistical analysis and processing of data. The measurement data were first tested for normality, and the data that met the normal distribution were expressed as mean ± standard deviation. One-way ANOVA was used to compare means for multiple independent groups of data that met normal distribution. When statistically significant results were obtained from the one-way ANOVA, pairwise comparisons of group means were performed. When variances were equal, the least significant difference t-test (LSD-t-test) was used to compare and analyze differences in means between groups. When variances were unequal, Dunnett's T3 test was used for statistical analysis. When variances did not meet normal distribution, the Kruskal-Wallis nonparametric test was used. P < 0.05 was considered statistically significant. Graphs were created using GraphPadPrism 6, Microsoft Office 365, and Photoshop CC 2019.

[0188] 5. Observation indicators

[0189] 1. General

[0190] The clinical manifestations of nephrotic syndrome include hyperlipidemia, severe edema, hyperproteinuria, hypoproteinemia, etc. When observing the SD rats with nephrotic syndrome induced by adriamycin, it is important to observe changes in the animal's mental state, body weight, food intake, water intake, coat color, and blood biochemistry.

[0191] 2. Urine protein / creatinine ratio

[0192] When collecting urine from SD rats, rat metabolic cages were used to collect the urine. The feces and urine of the rats were collected separately, and the changes in the 24-hour urine protein / creatinine ratio were detected.

[0193] 3. Organ Index

[0194] When the animals were collected, the liver and kidneys were removed and the excess water was absorbed with filter paper. The wet weight was then weighed to calculate the organ index (organ index = organ weight (g) / rat body weight (g) × 100%).

[0195] 4. Kidney wax staining

[0196] One-half of the kidney was fixed with 4% paraformaldehyde for 24-48 hours, then dehydrated in an automatic dehydrator and embedded in paraffin. Each kidney tissue was finally cut into 3 μm wax slices for subsequent experiments as follows:

[0197] H&E staining: Before staining, sections were baked at 65°C for 1.5 h to secure the tissue attachment. H&E staining was then performed as follows: dewaxing: xylene I for 10 min, xylene II for 10 min, and xylene III for 10 min; rehydration: 100% ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, 50% ethanol for 5 min, 20% ethanol for 5 min, and H2O for 5 min.

[0198] Hematoxylin staining: stain with hematoxylin for 15 minutes; color separation: separate with hydrochloric acid alcohol (0.1% hydrochloric acid / 75% ethanol) for a few seconds to make the cell nucleus turn blue, and immediately rinse with running water for a few minutes to wash off the excess stain.

[0199] Eosin staining: stain the cytoplasm with eosin solution for 5 minutes; dehydration and transparentization: 75% ethanol for 2 minutes, 85% ethanol for 2 minutes, 95% ethanol for 2 minutes, 100% ethanol for 2 minutes, 100% ethanol:xylene (1:1) for 2 minutes, xylene for 2 minutes; sealing and microscopic examination: sealing with neutral gum and microscopic examination.

[0200] 5. Blood biochemical index detection

[0201] When the animals were collected, rats were anesthetized by intraperitoneal injection, and blood was collected from the abdominal aorta using a coagulant tube. The collected blood was centrifuged at 3000 r / min for 15 min, and the serum was aspirated and stored at -80°C for later use.

[0202] Biochemical indicators BUN, Cr, ALB, ALT, and AST were measured using Nanjing Jiancheng kits according to the manufacturer's instructions. TC and TG were measured using a fully automatic biochemical indicator analyzer.

[0203] 6. WB experiment:

[0204] Animal Tissue Protein Extraction: 30 mg of kidney tissue was placed in a tissue homogenizer tube and 400 μL of tissue lysis buffer containing protease and phosphatase inhibitors was added (Roche tablets should be used at 1 tablet / 10 mL). After high-speed homogenization, centrifuge at 12,000 rpm for 15 minutes. The supernatant was collected and the protein concentration of the sample was determined by the BCA assay. After adjusting the concentration of each sample to a consistent level, 5x protein loading buffer was added and the sample was denatured at 100°C for 10 minutes. The sample was then stored at -80°C.

[0205] Use Yazyme premix to prepare polyacrylamide gel, load 20-30ug of sample, run the sample through the stacking gel at 80V, then adjust the voltage to 100V to run the sample through the entire gel to complete the SDS-PAGE gel electrophoresis operation; secondly, perform the membrane transfer operation (PVDF membrane, pre-wetted in methanol for 5 minutes in advance), the transfer conditions are: 300mA, 60min; after the transfer is completed, block with 5% skim milk for 2h, and incubate the primary antibody in a refrigerator at 4℃ overnight according to the corresponding band; the next day, wash away the unbound primary antibody with TBST washing solution (wash 3 times, 5min each time); incubate with secondary antibody at 37℃ for 2h, and wash away the unbound secondary antibody with TBST washing solution (wash 3 times, 5min each time); ECL luminescent liquid is used as a substrate to expose the target band, and the results are analyzed, recorded and statistically analyzed.

[0206] 6. Experimental Results

[0207] Figure 13 The graph shows the changes in body weight of SD rats with nephrotic syndrome induced by adriamycin in each group.

[0208] Depend on Figure 13 It can be seen that compared with the blank group, the body weight of the rats in the model group was significantly reduced, but there was no significant difference between the drug-treated group and the model group.

[0209] Figure 14 This figure shows the changes in urine protein / creatinine ratio in SD rats with nephrotic syndrome induced by adriamycin in each group.

[0210] from Figure 14 It can be seen that three weeks after the model was established by a single injection of doxorubicin through the tail vein, the urine protein / creatinine ratio of each group increased compared with the blank group, and there was a significant difference compared with the blank group (P<0.01). Drug administration began three weeks after modeling. Compared with the model group, the urine protein / creatinine ratio of the LXHK-L, LXHK-M, LXHK-H groups and the positive drug group decreased significantly after four weeks of oral gavage, and the difference was statistically significant (P<0.01). However, the urine protein / creatinine ratio of the TG group did not decrease significantly until five weeks of oral gavage (P<0.05). The onset time of the LXHK-L, LXHK-M, and LXHK-H groups in reducing the urine protein / creatinine ratio was earlier than that of the TG group, indicating that the effect of the tripterygium wilfordii polyglycosides combination on reducing the urine protein / creatinine ratio was better than that of tripterygium wilfordii polyglycosides.

[0211] Figure 15 The intervention renal index diagrams of SD rats with nephrotic syndrome induced by adriamycin in each group; Figure 16 The liver index of SD rats with nephrotic syndrome induced by adriamycin in each group was shown.

[0212] from Figure 15 It can be seen that the renal index of the model group was significantly increased compared with the blank group, while the renal index of each drug-treated group was decreased compared with the model group, and the difference was significant (P<0.01).

[0213] from Figure 16 It can be seen that the liver index of the model group was significantly increased compared with the blank group, while the liver index of the TG group was decreased compared with the model group, and the difference was significant (P<0.05). The liver index of the LXHK-M and LXHK-H groups was decreased compared with the model group, and the difference was extremely significant (P<0.01).

[0214] Figure 17 These are the spleen index graphs of SD rats with nephrotic syndrome induced by adriamycin in each group; Figure 18 Figure 3 is the cardiac index diagram of each group of SD rats with nephrotic syndrome induced by doxorubicin.

[0215] from Figure 17It can be seen that compared with the blank group, the spleen index of the model group decreased to a certain extent; compared with the model group, the spleen index of the TG group recovered to a certain extent after treatment, and the recovery effect of the spleen index in the LXHK-L group, LXHK-M group, and LXHK-H group was more obvious.

[0216] from Figure 18 It can be seen that compared with the blank group, the cardiac index of the model group increased to a certain extent; compared with the model group, the cardiac index of the TG group was further increased after medication, while the cardiac index of the LXHK-L group and the LXHK-M group was further decreased after medication.

[0217] Figure 19 The kidney tissue pathological images of SD rats with nephrotic syndrome induced by adriamycin in each group (200X).

[0218] Depend on Figure 19 Compared with the blank group, the model group showed severe renal tubular damage, including tubular dilatation with granular degeneration, partial vacuolar degeneration of renal tubular epithelial cells, brush border loss, multiple protein casts, and partial inflammatory cell infiltration. Glomeruli showed varying degrees of atrophy and deformation. After administration, the renal tissue of rats in the LXHK-L, LXHK-M, LXHK-H, and TG groups all showed varying degrees of relief, indicating that the tripterygium wilfordii polyglycosides combination has a certain effect in alleviating renal tissue damage caused by doxorubicin, and its effect is comparable to that of TG.

[0219] Figure 20-22 The statistical diagram of the changes in renal function and serum biochemistry of SD rats induced by adriamycin in each group, among which, Figure 20 This is a statistical chart of changes in serum urea nitrogen. Figure 21 This is a statistical chart of changes in serum creatinine. Figure 22 This is a statistical chart of serum albumin changes.

[0220] Depend on Figure 20 It can be seen that after modeling, serum urea nitrogen increased significantly, and after medication, the serum urea nitrogen values of each medication group decreased significantly (P<0.01), among which the decrease in serum urea nitrogen values of the LXHK-M group and the LXHK-H group was better than that of the TG group.

[0221] Depend on Figure 21 It can be seen that after modeling, the serum creatinine value increased, while the serum creatinine value of each medication group was significantly reduced after medication, and the difference was statistically significant.

[0222] Depend on Figure 22 It can be seen that after modeling, the serum albumin level in the model group decreased significantly (P<0.01), and after administration of the TG group and the LXHK-M and LXHK-H groups, the serum albumin values recovered (P<0.05).

[0223] Figure 23-24It is a statistical chart of changes in serum total cholesterol (TC) and blood lipid index triglyceride (TG).

[0224] Depend on Figure 23 It can be seen that after modeling, the serum total cholesterol showed a significant increase compared with the blank group, and after administration, the serum total cholesterol in the TG group was significantly decreased compared with the model group, and the difference was significant (P<0.05). The serum total cholesterol in the LXHK-L, LXHK-M, and LXHK-H groups was significantly decreased compared with the model group, and the difference was extremely significant (P<0.01), indicating that the intervention of the tripterygium wilfordii polyglycosides combination can significantly reduce the serum total cholesterol content, and its effect is better than that of tripterygium wilfordii polyglycosides.

[0225] Depend on Figure 24 It can be seen that after modeling, the triglyceride content increased significantly, while each medication group was able to effectively reduce the triglyceride content.

[0226] Figure 25 Western blot images of the changes of Cleave-caspase3 and Bax proteins in renal tissue of rats with nephrotic syndrome induced by doxorubicin in each group.

[0227] Depend on Figure 25 It can be seen that the total apoptosis execution protein Cleave-caspase3 and the pro-apoptotic protein Bax in the model group were significantly increased, while the expression levels of Cleave-caspase3 and Bax in each drug-treated group decreased, indicating that the combination of TG and tripterygium wilfordii polyglycosides can reduce the production of apoptotic proteins Cleave-caspase3 and Bax in renal tissue cells. The protective effect of TG and tripterygium wilfordii polyglycosides on renal tissue may be achieved by reducing the occurrence of apoptosis.

[0228] In summary, in rats with doxorubicin-induced nephrotic syndrome, the tripterygium wilfordii polyglycosides combination can reduce proteinuria, improve renal function, lower blood lipids, reduce cell apoptosis, and alleviate renal pathological damage. Furthermore, compared with tripterygium wilfordii polyglycosides, the tripterygium wilfordii polyglycosides combination has a better inhibitory effect on the kidney, liver, and heart toxicity that occurs during the treatment of nephrotic syndrome.

[0229] Experiment 3: Animal Pharmacodynamics of Tripterygium Wilfordii Polyglycosides Composition in Treating Psoriasis

[0230] 1. Experimental Animals

[0231] The experimental animals used in the experiment were SPF - level healthy male BalB / c mice, purchased from the Guangdong Provincial Medical Experimental Animal Center, with license numbers: SCXK(Guangdong)2013 - 0002, SCXK(Guangdong)2018 - 0002. The feeding environment for the experimental animals was as follows: maintained in a 12 - hour cycle under light / dark conditions (lights on: 07:00 - 19:00), controlling the temperature (20 ± 2 °C), humidity (50 ± 10 %), ventilation, with more than 10 air changes per hour (all fresh air systems), and allowing free access to standard rodent food and tap water. All animal experiment protocols were carried out in accordance with the "Regulations on the Administration of Experimental Animals" promulgated by the National Science and Technology Commission and approved by the Experimental Animal Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine (Experimental Animal Ethics Number: 2021018).

[0232] II. Drug Preparation

[0233] Appropriately suspend Tripterygium glycosides in 0.5% sodium carboxymethylcellulose (CMC - Na) solution respectively to prepare Tripterygium glycosides solutions with concentrations of 0.91 mg / mL and 8.19 mg / mL for standby use.

[0234] Add the Artemisia annua extract to the above - mentioned Tripterygium glycosides solution to prepare the Tripterygium glycosides composition solution, and keep it for use after ultrasonic suspension. Specifically, add an appropriate amount of Kunxining (AE - n1) prepared in Example 1 to the 0.91 mg / mL Tripterygium glycosides solution according to the concentration of Artemisia annua extract in the solution being 0.11 mg / ml to prepare a low - dose Tripterygium glycosides composition solution; add an appropriate amount of Kunxining (AE - n1) prepared in Example 1 to the 8.19 mg / mL Tripterygium glycosides solution according to the concentration of Artemisia annua extract in the solution being 1 mg / ml to prepare a high - dose Tripterygium glycosides composition solution.

[0235] Mixture solution of Tripterygium glycosides and artemisinin: Weigh an appropriate amount of artemisinin and suspend it in the 8.19 mg / mL Tripterygium glycosides solution to obtain a mixture solution, and the mixture solution contains 0.5 mg / mL of artemisinin.

[0236] Mixture solution of Tripterygium glycosides and artemisinene: Weigh an appropriate amount of artemisinene and suspend it in the 8.19 mg / mL Tripterygium glycosides solution to obtain a mixture solution, and the mixture solution contains 0.5 mg / mL of artemisinene.

[0237] Methotrexate: The dosing dose for mice is 1 mg / kg / day, and it is configured into a 0.1 mg / mL methotrexate solution using 0.5% CMC - Na solution.

[0238] III. Grouping, Model Establishment and Administration Methods

[0239] SPF healthy female BalB / c mice were adaptively fed for 3 days and then randomly divided into 8 groups: blank group (control), model group (Model), tripterygium wilfordii polyglycosides group (TG), tripterygium wilfordii polyglycosides + artemisinin group (TG+ART), tripterygium wilfordii polyglycosides + artemisinin group (TG+AT), low-dose tripterygium wilfordii polyglycosides combination group (LXHK-L), high-dose tripterygium wilfordii polyglycosides combination group (LXHK-H) and methotrexate group (MTX), with 4 mice in each group.

[0240] Except for the blank group, mice in each group received 62.5 mg of imiquimod cream (IMQ) applied to their backs at the same time daily for 8 consecutive days. The drug-treated group was also given the drug by gavage once daily for 8 consecutive days. The dosing schedule for each group is as follows:

[0241] Blank group (control): The mice were given an equal amount of CMC-Na solution by gavage according to their body weight every day;

[0242] Model group: The mice were given an equal amount of CMC-Na solution by gavage every day according to their body weight;

[0243] Tripterygium wilfordii polyglycosides group (TG): 81.9 mg / kg / day of Tripterygium wilfordii polyglycosides solution was given orally;

[0244] Tripterygium wilfordii polyglycosides + artemisinin group (TG+ART): 81.9 mg / kg / day of tripterygium wilfordii polyglycosides (9 times the adult clinical dose) and 5 mg / kg / day of artemisinin were administered by oral gavage.

[0245] Tripterygium wilfordii glycosides + artemisinin group (TG+AT): Administer a mixed solution of tripterygium wilfordii glycosides and artemisinin by oral gavage at a dose of 81.9 mg / kg / day of tripterygium wilfordii glycosides and 5 mg / kg / day of artemisinin.

[0246] The low-dose tripterygium wilfordii polyglycosides combination group (LXHK-L) was given a low-dose tripterygium wilfordii polyglycosides combination solution by oral gavage at 9.1 mg / kg / day (1 times the adult clinical dose) and 1.1 mg / kg / day of Artemisia annua extract.

[0247] High-dose tripterygium wilfordii polyglycosides combination group (LXHK-H): Administered with high-dose tripterygium wilfordii polyglycosides combination solution by oral gavage at 81.9 mg / kg / day of tripterygium wilfordii polyglycosides and 10 mg / kg / day of Artemisia annua extract;

[0248] Methotrexate group (MTX): methotrexate solution was administered orally at a dose of 1 mg / kg / day (calculated based on the adult clinical oral dose).

[0249] 4. Statistical Methods

[0250] GraphPad Prism 8.0.2 was used for statistical analysis and processing of data. The measurement data were first tested for normality, and the data that met the normal distribution were expressed as mean ± standard deviation. One-way ANOVA was used to compare means for multiple independent groups of data that met normal distribution. When statistically significant results were obtained in the one-way ANOVA, pairwise comparisons of group means were performed. When variances were equal, the least significant difference t-test (LSD-t-test) was used to compare and analyze differences in group means. When variances were unequal, Dunnett's T3 test was used for statistical analysis. When variances did not meet normal distribution, the Kruskal-Wallis nonparametric test was used. P < 0.05 was considered statistically significant. Graphs were generated using GraphPadPrism 8.0.2, Microsoft Office 365, and Photoshop CC 2019.

[0251] 5. Observation indicators

[0252] 1. General

[0253] The mental state of the mice was observed and their body weight was recorded.

[0254] 2. Liver function test

[0255] The eyeballs of mice were removed for blood collection. The blood was collected in yellow coagulant tubes, kept at 4°C for 2 h, and centrifuged at 3000 rpm for 15 min at room temperature. The supernatant was collected and serum ALT and AST were detected according to the method indicated in the kit instructions.

[0256] 3. Organ index

[0257] After the experiment, the spleen, kidneys, liver, ovaries, and uterus of the mice were removed and excess water was removed using filter paper. The wet weight was then weighed and the organ index was calculated: organ index = organ weight (g) / mouse body weight (g) × 100%.

[0258] 4. Skin lesions

[0259] The skin lesions of the mice were observed for epidermal scales, lesion texture, lesion thickness, erythema and punctate hemorrhage.

[0260] 5. Assessment of skin lesion severity

[0261] The Psoriasis Area and Severity Index (PASI) was used to assess the severity of psoriasis lesions in each group of mice before and at the end of treatment. The PASI score includes erythema, epidermal scaling, and skin thickness at the lesion site. The PASI scoring criteria are shown in Table 1. The total score is the sum of erythema, scaling, and skin thickness, based on the lesion severity score (0 to 4 points).

[0262] Table 1 PASI scoring criteria

[0263] score Severity erythema Epidermal squama Skin thickness 0 Asymptomatic none none flush with normal skin 1 Mild Pale red spots Epidermal squama Slightly higher than normal skin 2 Moderate Red Plate Covered with flaky epidermal scales Moderate bulge 3 severe Deep red patches Thick, layered epidermal scales Obvious bulge 4 Extremely severe Very deep red spots Thick, layered epidermal scales Obvious thickening of the bulge

[0264] 6. Measure the thickness of the skin lesions

[0265] Use Image-Pro Plus software to calibrate the ruler and click Measure Distances. Randomly select 5 locations in each photo to measure the epidermal thickness (the distance from the stratum corneum to the basement membrane zone). The average of the 5 measurements represents the visual field measurement data for subsequent statistical analysis.

[0266] 7. Observe the distribution of skin blood vessels at the lesion site

[0267] After the experiment, the back skin was cut off to observe the distribution of subcutaneous blood vessels in the lesion area of the mice.

[0268] 8. HE staining of skin tissue

[0269] The mouse skin lesion tissue was separated and 0.5 cm 2 Skin tissue was fixed with 4% paraformaldehyde for 24-48 h, dehydrated, embedded in paraffin, cut into 4 μm wax slices, and the pathological changes of skin lesion tissue were observed after HE staining.

[0270] HE staining process:

[0271] ① Before staining, bake the sections at 65°C for 1 hour to secure the tissue attachment; then proceed with the H&E staining procedure: dewaxing: xylene I for 10 minutes, xylene II for 10 minutes; rehydration: 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and H2O for 3 minutes.

[0272] ②Hematoxylin staining: stain with hematoxylin for 5 minutes; color separation: separate with hydrochloric acid alcohol (0.1% hydrochloric acid / 75% ethanol) for a few seconds to make the cell nucleus turn blue, and immediately rinse with running water for a few minutes to wash off the excess stain.

[0273] ③ Eosin staining: stain the cytoplasm with eosin solution for 3 min; dehydration and transparentization: 80% ethanol for 20 s, 90% ethanol for 20 s, 95% ethanol I for 1 min, 95% ethanol II for 1 min, 100% ethanol I for 2 min, 100% ethanol II for 2 min, xylene I for 2 min, xylene II for 2 min; mounting and microscopic examination: mounting with neutral gum and microscopic examination.

[0274] 9. Immunohistochemical staining to observe the expression of PCNA in the skin of the lesion

[0275] Immunohistochemistry staining process:

[0276] ① Dewaxing and hydration: Bake the sections in a 60°C oven for approximately 1 hour. Immediately soak in xylene for 10 minutes twice. Then, soak in a gradient of alcohol (100% ethanol → 100% ethanol → 95% ethanol → 95% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → 50% ethanol → 20% ethanol) for 5 minutes each. Remove the sections and rinse with distilled water before placing them in PBS buffer.

[0277] ② Antigen retrieval: Soak the tissue sections in a retrieval box and inject the diluted citric acid antigen retrieval solution. Heat in a microwave oven at medium-high heat (80-90°C) for 8 minutes, then stop heating for 8 minutes. Heat again at medium-low heat (50-60°C) for 8 minutes. Remove from the heat retrieval box, open the lid, and cool to room temperature. Rinse with PBS buffer three times for 5 minutes.

[0278] ③ Block endogenous peroxidase: wipe off excess PBS buffer on the slice, place it in an incubation box, add 100 μl of 3% H2O2 solution, let it stand at 37°C for 10 minutes, and rinse with PBS buffer 3 times for 3 minutes.

[0279] ④ Serum blocking: Add normal goat serum working solution for blocking into the histochemical circle and incubate at room temperature for 15 minutes.

[0280] ⑤ Primary antibody incubation: Gently shake off the blocking solution, do not wash, add an appropriate amount of prepared primary antibody solution (Note: the primary antibody solution is 5% bovine serum albumin solution with PCNA anti-rabbit primary antibody added at a ratio of 1:4000), place in a moisturizing box, and incubate at 4°C overnight.

[0281] ⑥ Secondary antibody incubation: The next day, warm the sections in a 37°C incubator for approximately 30 minutes. Rinse with PBS buffer three times for 5 minutes. Add commercial anti-rabbit secondary antibody solution and incubate at room temperature for 15 minutes. Rinse with PBS buffer three times for 3 minutes.

[0282] ⑦Add an appropriate amount of commercial HRP horseradish enzyme working solution, incubate at room temperature for 15 minutes, and rinse with PBS buffer 3 times for 3 minutes.

[0283] DAB color development: Add the prepared DAB color development solution (freshly prepared and used) on the tissue, control the color development time under a microscope, and when brown-yellow color appears, rinse with tap water to stop the color development.

[0284] ⑧Restain the cell nucleus: add hematoxylin solution for about 1 minute, rinse with tap water, turn blue in 0.1% ammonia solution for about 60 seconds, and rinse with double distilled water.

[0285] ⑨ After dehydration, transparency, and sealing, observe under an optical microscope, and collect and analyze images for evaluation.

[0286] 6. Experimental Results

[0287] Figure 26 The graph shows the daily changes in body weight of mice in each group.

[0288] Depend on Figure 26 It can be seen that compared with the blank group, the body weight of the mice in the model group was significantly reduced, but there was no significant change in the body weight of the drug-treated groups compared with the model group.

[0289] Figure 27 is the spleen index of mice in each group.

[0290] Depend on Figure 27 It can be seen that compared with the blank group, the spleen index of the model group mice was significantly increased, proving that its pathogenesis is related to immunity; compared with the model group, the spleen index of the LXHK-H group showed a downward trend.

[0291] Figure 28 The skin lesions of mice in each group.

[0292] Depend on Figure 28 As can be seen, the skin of normal mice remained unchanged throughout the experiment. Mice in the model group began to develop mild erythema and skin infiltration and hypertrophy on day 2. Over time, the erythema deepened, the infiltration and hypertrophy became more pronounced, and flaky scales appeared. The skin lesions were most severe on days 6 and 7. The LXHK-H and LXHK-L groups showed significant reductions in erythema, scaling, and hypertrophy, and the severity of skin lesions was alleviated. The improvement in the LXHK-H group was more pronounced than in the LXHK-L group. Furthermore, compared with the model group, the severity of skin lesions in the LXHK-L and LXHK-H groups was comparable to that in the TG group, indicating that the tripterygium wilfordii polyglycosides combination and TG are equally effective in improving skin lesions in psoriasis mice.

[0293] Figure 29 、 30 , 31, and 32 are the scales, thickness, erythema and cumulative PASI scores of the skin lesions of mice in each group, respectively.

[0294] Depend on Figure 29-32It can be seen that except for the blank group, the PASI scores of mice in each group increased, and the score in the model group increased most significantly. The scores of the LXHK-H group and the MTX group showed a downward trend from the 8th day; the scores of the other groups continued to increase.

[0295] Figure 33 is the average thickness of the skin at the lesion site of mice in each group.

[0296] Depend on Figure 33 It can be seen that the epidermal thickness of the LXHK-H, LXHK-L and TG groups was at the same level, and there were statistical differences compared with the model group (P<0.01), indicating that the pharmacological effects of the tripterygium wilfordii polyglycosides combination and TG in reducing the skin thickness of psoriasis mice were consistent.

[0297] Figure 34 The distribution of subcutaneous blood vessels in the lesion area of mice in each group.

[0298] Depend on Figure 34 It can be seen that compared with the blank group mice, the number of blood vessels in the skin tissue of the model group increased significantly, and the vascular tortuosity increased; compared with the model group, except for the MTX group with a larger number of blood vessels and tortuosity, the number of blood vessels in the mice in each drug-treated group decreased, and the tortuosity was significantly improved.

[0299] Figure 35 The following are the pathological changes of the skin lesions on the back of mice in each group (HE staining, 200X).

[0300] Depend on Figure 35 As can be seen, in the blank group, there was no significant epidermal hyperplasia, a thin stratum corneum, flat epidermal projections, a small amount of chronic inflammatory cell infiltration in the dermis, and no significant vasodilation. In the model group, there was epidermal hyperplasia and hyperkeratosis, with numerous neutrophil infiltrations in the epidermis and stratum corneum, the stratum granulosum disappeared or became thinner, vasodilation in the superficial dermis, and scattered or focal infiltrations of chronic inflammatory cells (Note: In the figure, green arrows: microabscesses formed by neutrophils aggregated in the stratum corneum; yellow arrows: hyperkeratosis; orange arrows: epidermal thickening; blue arrows: acanthosis; black arrows: disappearance of the stratum granulosum; red arrows: mononuclear or multinuclear cell infiltration in the dermis; gray arrows: capillary dilation). Compared with the model group, the degree of epidermal hyperplasia and parakeratosis in all treatment groups was significantly reduced, the number of neutrophils in the epidermis and stratum corneum, and the number of inflammatory cells infiltrating the superficial dermis decreased, and the dilated blood vessels in the dermis decreased. Compared with the model group, the degree of lesion reduction in the LXHK-H and LXHK-L groups was comparable to that in the TG group, indicating that the pharmacological effects of the tripterygium wilfordii polyglycosides composition and TG in alleviating pathological changes in the skin of psoriasis mice were consistent.

[0301] Figure 36 The expression of PCNA in the skin tissue of mice in each group.

[0302] Depend on Figure 36 It can be seen that compared with the blank group, the PCNA level in the model group was significantly increased; compared with the model group, the PCNA expression levels in each drug-treated group were decreased, among which the expression levels in the TG group and the LXHK-H group decreased most significantly.

[0303] Figure 37 、 38 , 39 are the changes in liver function and liver index of mice in each group, respectively.

[0304] Depend on Figure 37 It can be seen that compared with the blank group, the ALT level in the model group showed an upward trend; compared with the model group, the ALT levels in the TG group and the LXHK-L group decreased; the ALT levels in the TG+ART group, the TG+AT group and the LXHK-H group increased, and the ALT level in the MTX group increased significantly, indicating obvious hepatotoxicity.

[0305] Depend on Figure 38 It can be seen that compared with the blank group, the AST level in the model group showed an upward trend; compared with the model group, the AST in the TG group showed no significant change, but the AST in the LXHK-L group showed a downward trend after administration.

[0306] Depend on Figure 39 It can be seen that compared with the blank group, the liver index of the model group increased significantly, which was statistically significant; compared with the model group, except for the liver index of the MTX group which was equivalent to the model group, the liver index of each drug-treated group showed a downward trend.

[0307] Figure 40 The changes of kidney index in each group of mice.

[0308] Depend on Figure 40 It can be seen that compared with the blank group, the renal index of the model group increased significantly, which was statistically significant; compared with the model group, except for the renal index of the MTX group which was equivalent to the model group, the renal index of each drug-treated group showed a downward trend.

[0309] Figure 41 、 42 Shown are the organ indexes and morphological changes of the ovary and uterus of mice in each group.

[0310] Depend on Figure 41 、 42 It can be seen that compared with the blank group, the organ indexes of the ovary and uterus in the model group were significantly reduced, the volume of the ovary and uterus became smaller, and the morphology was atrophied; compared with the model group, the ovary and uterine indexes of the TG group were lower, while the ovary and uterine indexes of the TG+ART, TG+AT, LXHK-L, LXHK-H and MTX groups recovered to a certain extent after administration, and the morphology of the ovary and uterus also recovered significantly.

[0311] In summary, in mice with imiquimod-induced psoriasis, the tripterygium wilfordii polyglycosides combination was able to suppress the immune response to a certain extent, reduce inflammatory cell infiltration, decrease PCNA protein expression, alleviate the formation of scales and skin keratinization in the lesion area, reduce erythema, and reduce subcutaneous angiogenesis, with an effect comparable to that of tripterygium wilfordii polyglycosides. Furthermore, compared with tripterygium wilfordii polyglycosides, the tripterygium wilfordii polyglycosides combination also had a certain degree of inhibitory effect on the liver and reproductive system toxicity that occurs during the treatment of psoriasis.

Claims

1. Kun Xining, characterized by, Its active ingredients are artemisinin and artemisinin.

2. The Kunxining according to claim 1, characterized in that The Kunxining is an Artemisia annua extract rich in artemisinin and artemisinin, wherein the mass ratio of artemisinin to artemisinin in the Artemisia annua extract rich in artemisinin and artemisinin is (0.8-1.2):(0.8-1.2), and the mass ratio of artemisinin to artemisinin in the Artemisia annua extract rich in artemisinin and artemisinin is 85-95%.

3. The Kunxining according to claim 2, characterized in that The mass ratio of artemisinin to artemisinin in the artemisia annua extract rich in artemisinin and artemisinin is 1:1, and the mass ratio of artemisinin to artemisinin accounts for 90% of the artemisia annua extract rich in artemisinin and artemisinin.

4. Use of the Kunxining according to any one of claims 1 to 3 in the preparation of Artemisia annua preparations.

5. Use of the Kunxining according to any one of claims 1 to 3 in the preparation of a medicament for reducing the toxicity of tripterygium wilfordii polyglycosides on the female reproductive system.

6. Use of the Kunxining according to any one of claims 1 to 3 in the preparation of a drug for reducing the ovarian toxicity of tripterygium wilfordii polyglycosides.

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