Traditional Chinese medicine composition and application thereof in preparation of medicine for relieving renal fibrosis caused by ferroptosis

Abstract

The invention relates to a traditional Chinese medicine composition and application of the traditional Chinese medicine composition in preparation of a medicine for relieving renal fibrosis caused by ferroptosis. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 to 30 parts of radix salviae miltiorrhizae, 9 to 12 parts of radix et rhizoma rhei preparata, 8 to 18 parts of peach kernel, 5 to 15 parts of radix angelicae sinensis, 9 to 15 parts of radix achyranthis bidentatae, 4 to 10 parts of radix paeoniae rubra, 9 to 20 parts of herba dendrodii officinalis, 6 to 15 parts of fructus ligustri lucidi, 5 to 10 parts of fructus corni, 9 to 15 parts of herba centellae, 3 to 6 parts of radix notoginseng powder and 10 to 30 parts of radix astragali seu hedysari. Compared with the prior art, the traditional Chinese medicine composition provided by the invention can relieve kidney fibrosis caused by ferroptosis and reduce the score of kidney injury, and compared with a model group, the expression of GSH, TFR-1, SLC7A11 and GPX4 in the traditional Chinese medicine composition group is remarkably up-regulated, and the content of 4-HNE and MDA is remarkably reduced, so that the kidney injury caused by ferroptosis is relieved, and the kidney protection effect on patients with chronic kidney diseases is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition and its application in preparing a medicine for alleviating renal fibrosis caused by ferroptosis. Background Art

[0002] Renal fibrosis, characterized primarily by scarring due to excessive extracellular matrix deposition, is a common endpoint in various forms of chronic kidney disease (CKD). However, there are currently no anti-fibrotic therapies specifically targeting CKD. Renal fibrosis is primarily identified by tubular atrophy, chronic interstitial inflammation, fibrosis, and glomerular sclerosis. In recent years, numerous marketed drugs (e.g., renin-angiotensin system (RAS) inhibitors, sodium-glucose cotransporter SGLT-2 inhibitors, and angiotensin II receptor antagonists) have been developed to slow the progression of CKD.

[0003] Chronic kidney disease caused by diabetes is severely affected by ferroptosis. Ferroptosis is a regulated cell death triggered by oxidative stress and iron-dependent lipid peroxidation. Ferroptosis is regulated by metabolic signaling pathways, including GPX4 dysfunction, the cystine / glutamate antiporter system, lipid peroxidation, and insufficient iron metabolism. For example, in mice with renal fibrosis induced by unilateral ureteral obstruction (UUO), GPX4 expression was observed to decrease with prolonged obstruction, while the expression of 4-HNE, a lipid peroxidation marker, gradually increased. This suggests that during renal fibrosis, GPX4 downregulation leads to a weakened intracellular antioxidant capacity, an inability to effectively clear lipid peroxidation products, and thus triggers ferroptosis.

[0004] However, there are currently no drugs for treating kidney disease caused by ferroptosis, and it is impossible to improve the treatment effect of kidney disease caused by ferroptosis in a more targeted and high-quality manner. Summary of the Invention

[0005] The purpose of the present invention is to fill the gap in therapeutic drugs for targeted ferroptosis-induced kidney disease, thereby providing a Chinese medicine composition and its use in the preparation of drugs for alleviating renal fibrosis caused by ferroptosis.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a traditional Chinese medicine composition, which is composed of the following raw materials in parts by weight:

[0008] 15-30 parts of Salvia miltiorrhiza, 9-12 parts of processed rhubarb, 8-18 parts of peach kernel, 5-15 parts of Chinese angelica, 9-15 parts of Cyathula, 4-10 parts of red peony root, 9-20 parts of Dendrobium officinale, 6-15 parts of Ligustrum lucidum fruit, 5-10 parts of Cornus officinalis, 9-15 parts of Centella asiatica, 3-6 parts of Panax notoginseng powder, and 10-30 parts of Astragalus.

[0009] In some specific embodiments, the Chinese medicine composition is composed of the following raw materials in parts by weight:

[0010] Salvia miltiorrhiza 15g, processed rhubarb 9g, peach kernel 12g, Chinese angelica 12g, Achyranthes bidentata 15g, red peony root 9g, Dendrobium officinale 10g, Ligustrum lucidum fruit 12g, Cornus officinalis 10g, Centella asiatica 10g, Panax notoginseng powder 6g, Astragalus 30g.

[0011] The second technical solution of the present invention is to provide a use of the traditional Chinese medicine composition as described in one of the above technical solutions in the preparation of a drug for alleviating renal fibrosis caused by ferroptosis.

[0012] In some embodiments, the drug further comprises a pharmaceutical excipient.

[0013] In some specific embodiments, the pharmaceutical excipient is selected from any one or more of a filler, a disintegrant, a binder, and a lubricant.

[0014] In some specific embodiments, the dosage form of the drug is selected from any one of granules, tablets, powders, and capsules.

[0015] In some embodiments, the drug is a drug that increases the activity and / or expression of antioxidant-related proteins and / or small molecules.

[0016] In some specific embodiments, the antioxidant-related protein is selected from any one or more of GPX4 and SLC7A11, and the small molecule substance is GSH.

[0017] In some embodiments, the drug is a drug that increases the expression of proteins related to iron metabolism.

[0018] In some specific embodiments, the protein related to iron metabolism is TFR-1.

[0019] In some embodiments, the drug is a drug that reduces the level of lipid peroxidation end products.

[0020] In some specific embodiments, the lipid peroxidation end product is selected from any one or more of 4-HNE and MDA.

[0021] In some specific embodiments, when used, the effective amount of the Chinese medicine composition in the medicine is 5g·kg -1·d~30g·kg -1 ·d.

[0022] Traditional Chinese Medicine considers CKD to be classified as "edema," "difficulty with constipation," and "consumption," generally characterized as a combination of underlying deficiency and superficial excess. The underlying deficiency is the decline of lung, spleen, and kidney function, with renal failure being the underlying cause. The superficial excess includes pathogenic factors such as stasis, dampness, turbidity, and toxins, all accompanied by the formation of blood stasis. Disturbances in iron and lipid metabolism are common in CKD patients, and these disturbances create favorable conditions for the occurrence of ferroptosis.

[0023] In the Chinese medicine composition of the present invention,

[0024] Salvia miltiorrhiza and processed rhubarb are the main ingredients, promoting blood circulation and removing blood stasis, clearing the bowels and purging heat; peach kernel, angelica, and Chinese cyperus are the secondary ingredients, enhancing the blood circulation and blood stasis removal, nourishing blood and tendons, and guiding blood downward; red peony root, dendrobium officinale, glossy privet fruit, cornus officinalis, centella asiatica, and Panax notoginseng powder are the auxiliary ingredients, clearing heat and cooling blood, nourishing yin and clearing heat, tonifying the liver and kidneys, astringing spermatorrhea and stopping nocturnal emission, clearing heat and dampness, detoxifying and reducing swelling, removing blood stasis and stopping bleeding, promoting blood circulation and relieving pain; astragalus is the auxiliary ingredient, tonifying qi and raising yang, promoting diuresis and reducing swelling. The whole formula works together to promote blood circulation and remove blood stasis, replenish qi and nourish yin, clear heat and remove dampness, and tonify the liver and kidneys. It is suitable for blood stasis, qi and yin deficiency, internal dampness and heat, and liver and kidney deficiency.

[0025] The functions of each component are:

[0026] Salvia miltiorrhiza is cold in nature, and can promote blood circulation, remove blood stasis, relieve numbness and relieve pain, while also replenishing kidney qi.

[0027] Peach kernel and Achyranthes bidentata are neutral in nature and can replenish qi, tonify the kidneys, strengthen the waist, promote blood circulation and remove blood stasis.

[0028] Rhubarb is cold in nature and can clear away heat, drain turbidity and promote blood circulation.

[0029] Angelica sinensis is warm in nature and can replenish blood, promote blood circulation, remove blood stasis and relieve pain.

[0030] Red peony root is bitter and slightly cold in nature, and can promote blood circulation and relieve blood stasis; red peony root extract can reduce the production of reactive oxygen species (ROS), alleviate oxidative stress, inhibit the uptake of iron ions and lipid peroxidation, and thus improve renal function by inhibiting ferroptosis.

[0031] Dendrobium officinale is sweet and slightly cold in nature, nourishes yin, replenishes qi, and promotes salivation; the alkaloids in Dendrobium officinale have antioxidant properties, which can reduce oxidative stress and thus inhibit ferroptosis.

[0032] Ligustrum lucidum fruit is sweet and bitter in nature, and nourishes kidney yin; the oleanolic acid it contains has antioxidant effects, can scavenge oxygen free radicals, reduce oxidative stress, and improve kidney function.

[0033] Cornus officinalis is slightly warm in nature and nourishes the liver and kidneys; the cornus officinalis polysaccharides it contains have antioxidant effects and can reduce oxidative stress.

[0034] Centella asiatica is cold in nature and bitter in taste. It can promote blood circulation, remove blood stasis, promote dampness and reduce swelling, clear away heat and dampness, and regulate kidney function. The asiatica glycoside it contains has antioxidant effects, can reduce oxidative stress and alleviate ferroptosis.

[0035] Panax notoginseng powder is warm in nature, sweet and slightly bitter in taste, can disperse blood stasis and reduce swelling, regulate kidney blood flow, and thus improve kidney function. Among them, Panax notoginseng total saponins have antioxidant effects and can reduce oxidative stress.

[0036] Astragalus is slightly warm in nature, sweet in taste, and can replenish qi and raise yang. The astragalus polysaccharides in astragalus can protect the kidneys, resist oxidation, reduce oxidative stress, and thus inhibit ferroptosis.

[0037] The Chinese medicine composition of the present invention promotes blood circulation and removes blood stasis, uses both warming and cooling effects, and attacks and supplements at the same time. It can not only strengthen the body but also clear the bowels and eliminate turbidity, so that turbid air can be discharged without damaging the body, and it can nourish the body without retaining evil, thereby effectively improving renal fibrosis caused by ferroptosis.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The Chinese medicine composition or medicine provided by the present invention can alleviate renal fibrosis caused by ferroptosis, reduce renal injury score, and significantly increase the expression of GSH, TFR-1, SLC7A11, GPX4 and Nrf2, and Fe 2+ , 4-HNE, and MDA levels decreased significantly, thereby reducing kidney damage caused by ferroptosis and playing a kidney-protective role in patients with chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The effect of the Chinese medicine composition on the pathomorphology of renal tissue in SD rats with renal fibrosis (HE and Masson, x200).

[0041] Figure 2 This is the effect of the Chinese medicine composition on the expression levels of GPX4, SCL7A11, and TFR-1 proteins in the renal tissue of SD rats.

[0042] Figure 3 Effect of Chinese herbal medicine combination on the expression of GPX4 and Nrf2 proteins in renal tissue (x 200) DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0044] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0045] 1. Materials:

[0046] 1.1 Animals

[0047] Fifty SPF Sprague-Dawley rats, weighing 200 ± 20 g, were obtained from Shanghai Slake Animal Co., Ltd. (license number: SCXK(Shanghai)2013-0016). All rats were housed at the Experimental Animal Center of Shanghai Hospital of Traditional Chinese Medicine, affiliated to Shanghai University of Traditional Chinese Medicine. All rats were maintained at a temperature of (24 ± 2)°C, with a 12-h light / dark cycle (7:00 AM–7:00 PM daily), and had free access to standard chow and water. This experiment was approved by the Experimental Animal Ethics Committee of Shanghai Hospital of Traditional Chinese Medicine, approval number: 2024142. The experiments were conducted after one week of adaptive feeding.

[0048] 1.2 Medication

[0049] The Chinese medicine composition is composed of 15g of Danshen (Henan, batch number: 16022001), 9g of processed rhubarb (Henan, batch number: 16016114), 12g of peach kernel (Yunnan, batch number: 160132021), 12g of Chinese angelica (Gansu, batch number: 160212135), 15g of Chinese clematis (Henan, batch number: 160215126), 9g of red peony root (Hebei, batch number: 16018121), and 10g of Dendrobium officinale. (Zhejiang, batch number: 16020137), Ligustrum lucidum fruit 12g (Zhejiang, batch number: 16014007), Cornus officinalis 10g (Henan, batch number: 16018037), Centella asiatica 10g (Shaanxi, batch number: 16051018), Panax notoginseng powder 6g (Yunnan, batch number: 16042009), and Astragalus membranaceus 30g (Gansu, batch number: 16017014) were provided by Shanghai Kangqiao Chinese Medicine Pieces Co., Ltd. Two deputy chief pharmacists at Shanghai Hospital of Traditional Chinese Medicine independently conducted blinded identification to ensure drug source assurance. Losartan potassium tablets (100 mg / tablet, Hangzhou Merck Pharmaceutical Co., Ltd., batch number: J20130048)

[0050] After mixing the above-mentioned Chinese medicine raw materials, follow the general Chinese medicine decoction steps, soak for 30 minutes, high heat for 5 minutes, and low heat for 20-30 minutes. Then filter out the residue and concentrate it to about 300-600mL. Then add anhydrous ethanol so that anhydrous ethanol accounts for 70% of the total volume. First, add the Chinese medicine and anhydrous ethanol in a volume ratio of 1:1 to the bottle and let it stand for 2 hours. After 2 hours, add the remaining anhydrous ethanol to the bottle overnight. The next day, use filter paper and funnel to filter the Chinese medicine into a new narrow-mouth bottle, and then concentrate the filtered Chinese medicine in batches. The concentration is carried out in a water bath or directly on an asbestos net. The concentration is calculated using the volume of the remaining liquid. Through the above steps, a Chinese medicine compound concentrate can be effectively prepared, the active ingredients in the Chinese medicine can be extracted, and impurities can be removed.

[0051] 1.3 Reagents

[0052] Micro-reduced glutathione (GSH) kit (Nanjing Jiancheng Biotechnology Co., Ltd., batch number A006-2-1);

[0053] Hematoxylin-eosin (HE) (Shanghai Bio-Tech, batch number: C0105M);

[0054] Masson (Shanghai Biyuntian, batch number: C0189M);

[0055] BCA kit (Wuhan Sewell Biotechnology Co., Ltd., batch number: G2026);

[0056] Rat 4-HNE enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Youbiai, U96-1284E);

[0057] GAPDH (No.#4060, CST, 1:2000);

[0058] Glutathione peroxidase-4 (GPX4) (No.#59735, CST, 1:1000);

[0059] glutathione (GSH) (No. G4305, Seville);

[0060] Solute carrier family 7 member 11 (SCL7A11) (No.#23276, CST, 1:1000);

[0061] Transferrin receptor-1 (TFR-1) (No.#55487, CST, 1:1000);

[0062] Malondialdehyde (MDA) (Shanghai Biyuntian, batch number: S0131M);

[0063] Nrf2 (No.#12721, CST, 1:500);

[0064] Fe ion detection kit (Aibixin, abs580105).

[0065] 1.4 Instruments

[0066] BioTek Synergy2, Pathology microtome (LEICA, RM2016), Automatic imaging analysis system (ProteinSimple FluorChem M), Centrifuge (eppendorf, 5417R), Automatic biochemical analyzer (Hitachi 7080, Tokyo), Orthosteric microscope (Zeiss Axio Lab Al), Vertical electrophoresis tank (Bio-rad, 1658004), Electrophoresis instrument (Sevier, SVT-2), Membrane transfer instrument (Bio-rad, 1652100), Laser confocal microscope (Leica, TCS SP5).

[0067] Example 1

[0068] 1 Model preparation, grouping and administration

[0069] The chronic renal failure rat model was established according to the Platt method: 5 / 6 nephrectomy induced renal fibrosis model (reference: Ma SK, Joo S Y, Kim CS, et al. Increased Phosphorylation of PI3K / Akt / mTOR in the Obstructed Kidney of Rats with Unilateral Ureteral Obstruction [J]. Chonnam Medical Journal, 2013, 11(3): 108-112.), the sham operation group only peeled off the skin and muscle envelope in the kidney area during the operation, exposed the kidney, and then put the kidney into the wound and sutured the wound.

[0070] 60 SD rats were divided into a sham operation group of 10 and a model group of 50, and after 2 weeks, the Scr was measured by the internal canthus blood, and if there was a statistical difference between the Scr of the model group and the sham operation group, the modeling was successful. The model rats were randomly divided into a model group, a traditional Chinese medicine composition low-dose group, a traditional Chinese medicine composition high-dose group, a losartan potassium group and a control formula group, each group of 10, according to the conversion coefficient of 20 times of the adult dose of 60 kg, the losartan potassium group was given 33.3 g·kg -1 ·d, the traditional Chinese medicine composition group (low dose 10.5 g·kg -1 ·d, high dose 21 g·kg -1 ·d), and the control formula group was given 21 g·kg -1 ·d) by gavage; the sham operation group and the model group were given the same volume of normal saline, 1 mL / 100 g, once a day, and were fed for 9 weeks.

[0071] The comparative formula group was composed of: 15g of Danshen, 9g of processed rhubarb, 12g of peach kernel, 12g of angelica, and 15g of Achyranthes bidentata. The dosage of the comparative formula was 21g·kg -1 .

[0072] In this comparative formula, Danshen (Salvia miltiorrhiza) is the main herb, promoting blood circulation and removing blood stasis, relieving pain and promoting menstruation. Processed rhubarb and peach kernel are the auxiliary herbs. Processed rhubarb is a purgative, dispelling stasis and promoting menstruation, while peach kernel promotes blood circulation and removes blood stasis, moistening the intestines and promoting bowel movements. Angelica sinensis is the adjuvant, tonifying blood and promoting blood circulation, regulating menstruation and relieving pain. Achyranthes bidentata is the guiding herb, removing blood stasis and promoting menstruation and guiding blood downward.

[0073] 2. Sample collection and indicator detection

[0074] After drug intervention, rats in the sham-operated group were in good spirits, active, with neat fur, and gained weight. Rats in the model, losartan potassium, low-dose, and high-dose Chinese medicine combinations, as well as the control formula, showed poor mental states, matted fur, decreased activity, and varying degrees of weight loss.

[0075] The rats included in the statistics were 7 rats in the model group, 8 rats in the losartan potassium group, 8 rats in the low-dose Chinese medicine composition group, 9 rats in the high-dose Chinese medicine composition group, and 9 rats in the control formula group.

[0076] At the end of the 9th week of drug intervention, rats in each group were fasted and deprived of water for 12 hours before being sacrificed. The rats were placed in metabolic cages to collect urine for 24 hours at 4°C and 3000 r·min. -1 Centrifuge for 5 minutes and send to the laboratory for testing immediately. -1 Intraperitoneal anesthesia, blood was collected from the abdominal aorta, and the temperature was kept at 4°C, 3000 r·min. -1 Centrifuge for 10 minutes to separate the rat serum for later use. Simultaneously, the remaining left kidney of the rat was quickly dissected and removed. Washed with saline, the kidney was placed on dry ice to remove blood stains, and the renal capsule and adipose tissue were removed. The kidney was then cut longitudinally into two halves. One half of the left kidney tissue was fixed in 4% paraformaldehyde; the other half was placed in a cryovial and quickly frozen in liquid nitrogen for measurement of relevant indicators.

[0077] 3. Detection of serum creatinine (Scr), urea nitrogen (Bun), and 24-hour urine protein (24h-Upro) in rats

[0078] The concentrations of serum creatinine, urea nitrogen and urine protein in rats of each group were measured by automatic biochemical analyzer.

[0079] 24h-Upro = urine protein concentration * total urine volume in 24 hours.

[0080] Statistical analysis was performed using GraphPad Prism 10.3.0. One-way ANOVA was used to compare differences between groups. If there was a statistically significant difference (P < 0.05), the Fisher LSD test was used as a follow-up test after the ANOVA.

[0081] As shown in Table 1, compared with the sham-operated group, the model group showed significant increases in Scr, Bun, and 24h-Upro levels (P < 0.01). Compared with the model group, the Scr, Bun, and 24h-Upro levels of SD rats in the losartan potassium group and the low- and high-dose Chinese medicine combination groups were significantly decreased (P < 0.05).

[0082] Table 1 Effects of Chinese herbal medicine combination on renal function in SD rats with renal fibrosis

[0083]

[0084] Note: Compared with the sham operation group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P < 0.01.

[0085] As can be seen from Table 1, the levels of Scr, Bun and 24-Pro of the Chinese medicine composition of the present invention are better than those of the control formula, showing better improvement in renal function.

[0086] Example 2

[0087] Based on the specimens collected in Example 1, renal tissue morphology HE staining and Masson staining were performed.

[0088] Renal tissue samples were fixed in 4% paraformaldehyde and cut into thin sections. Sections were stained with hematoxylin and eosin and Masson's staining to assess the extent of tubular atrophy and renal fibrosis. Five non-overlapping tubular interstitial fields were selected at 200x magnification and examined for renal pathological changes using an upright light microscope. The results were averaged. Positive staining areas were calculated using Image Pro Plus 7.0.

[0089] Statistical analysis was performed using GraphPad Prism 10.3.0. One-way ANOVA was used to compare differences between groups. If there was a statistically significant difference (P < 0.05), the Fisher LSD test was used as a follow-up test after the ANOVA.

[0090] like Figure 1As shown in Table 2, compared with the sham-operated group, HE staining revealed that the model group exhibited mesangial matrix proliferation, tubular atrophy, focal, staged, moderate to severe glomerular sclerosis and enlargement, marked tubular dilatation, glomerular adhesions, tubular rupture, significant cystic expansion, vacuolar and granular degeneration of epithelial cells, increased fibrotic renal tissue, decreased renal interstitial blood vessels, and inflammatory cell infiltration (P < 0.01). Compared with the model group, the losartan potassium group and the low- and high-dose Chinese medicine combination groups showed a small amount of inflammatory cell infiltration in the renal interstitium, mild glomerular deformation, and significantly improved fibrosis compared with the model group.

[0091] Compared with the sham-operated group, Masson staining revealed increased glomerular volume, interstitial inflammatory cell infiltration, increased mesangial cells, thickened basement membranes, and significantly increased collagen fiber deposition in the model group. Compared with the model group, the losartan potassium group and the low- and high-dose Chinese medicine combination groups showed a significant decrease in collagen fiber area.

[0092] Table 2 Effects of the Chinese herbal composition on renal tissue injury score and positive staining area in rats with renal fibrosis

[0093]

[0094] Note: Compared with the sham operation group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P < 0.01.

[0095] As can be seen from Table 2, the Chinese medicine composition of the present invention is superior to the control formula in terms of the levels of glomerular injury, tubular injury and positive staining area, showing more excellent improvement in renal function.

[0096] Example 3

[0097] Based on the samples collected in Example 1, the rat kidney tissue Fe 2+ , MDA, GSH content

[0098] The rat kidney tissue was obtained and ground with pre-cooled PBS to prepare a 10% tissue homogenate. The supernatant was collected by centrifugation and the supernatant was determined according to the Fe 2+ , MDA, and GSH kit instructions: BCA was used to determine protein concentration, and colorimetry was used to determine iron content. The MDA absorbance A was determined at 535 nm using the TBA method on a microplate reader. Simultaneously, the OD value was measured at 412 nm, representing GSH activity.

[0099] Based on the samples collected in Example 1, the 4-HNE level in rat kidney tissue was measured by ELISA.

[0100] The rat kidney tissue homogenate supernatant was taken, and the ELISA kit instructions were strictly followed according to the experimental steps.

[0101] Statistical analysis was performed using GraphPad Prism 10.3.0. All numerical values were expressed as One-way ANOVA was used for comparison of differences between groups. If there was a statistically significant difference (P<0.05), the subsequent test of analysis of variance used Fisher LSD test.

[0102] As shown in Table 3, compared with the sham operation group, the Fe 2+ , MDA and 4-HNE in the model group were significantly increased, and the GSH activity was significantly decreased, with statistically significant difference (P<0.01). Compared with the model group, the Fe 2+ , MDA and 4-HNE in the losartan potassium group and the low and high dose groups of the traditional Chinese medicine composition were significantly reduced, and the GSH activity in the high dose group of the traditional Chinese medicine composition was increased, with statistically significant difference (P<0.05, P<0.01).

[0103] Table 3 Effect of traditional Chinese medicine composition on MDA, GSH and 4-HNE content in kidney tissue of rats with renal fibrosis

[0104]

[0105] Note: compared with the sham operation group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0106] Example 4

[0107] Based on the specimens collected in Example 1, the levels of GPX4, SCL7A11 and TFR-1 in rat kidney tissue were detected by WB

[0108] According to the kit instructions, the protein of residual kidney tissue of rats was extracted, homogenized on ice, and centrifuged to take the supernatant. After protein quantification, denaturation was performed in boiling water bath at 95℃ for 10 minutes, 10% gel electrophoresis was performed according to the SDS-RAGE gel instructions, and transfer to PVDF membrane for 90 minutes. After blocking with skim milk at room temperature for 30 minutes, TFR-1 antibody, SLC7A11 antibody and GPX4 antibody were added respectively, and incubated overnight at 4℃. The next day, TBST was washed for three times, the secondary antibody was diluted at 1:5000, and incubated at room temperature for 1 hour. Development and exposure. Image J calculates the gray value ratio of the target band to the internal reference GAPDH, which represents the relative expression of the target protein.

[0109] Statistical analysis was performed using GraphPad Prism 10.3.0. All values are expressed as mean ± SD. One-way ANOVA was used for comparison of differences between groups. If there was a statistically significant difference (P < 0.05), the subsequent test of analysis of variance used Fisher LSD test.

[0110] As shown in Table 4, compared with the sham operation group, the expression of TFR-1, SLC7A11 and GPX4 proteins in the kidney tissues of the model group was significantly reduced (P < 0.01); compared with the model group, the expression of TFR-1, SLC7A11 and GPX4 proteins in the rats of the losartan potassium group and the low and high dose groups of the traditional Chinese medicine composition was significantly increased, and the difference was statistically significant (P < 0.05, P < 0.01). Figure 2 Table 4 Comparison of GPX4, SCL7A11 and TFR-1 protein expression in kidney tissues of rats in each group

[0111]

[0112]

[0113]

[0114] Note: compared with the sham operation group, * P < 0.05, ** P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01.

[0115] Example 5

[0116] Based on the specimens collected in Example 1, the levels of F4 / 80 and Nrf2 in the kidney tissues of rats were detected by immunofluorescence

[0117] The kidneys were fixed with 4% paraformaldehyde, and 5 μm kidney tissue sections were deparaffinized in xylene and rehydrated using graded ethanol. Antigen retrieval was performed by microwave for 10 minutes, and to block non-specific binding, the sections were incubated with PBS containing 20% goat serum for 30 minutes. Kidney tissue sections were incubated overnight at 4°C with monoclonal antibodies against GPX4 and Nrf2, respectively, and nuclear staining was performed using DAPI. The sections were imaged using a laser confocal microscope.

[0118] Compared with the sham operation group, the expression of GPX4 and Nrf2 in the kidney tissues of the model group was significantly reduced (P < 0.01); compared with the model group, the expression of GPX4 and Nrf2 in the rats of the losartan potassium group and the low and high dose groups of the traditional Chinese medicine composition was significantly increased, and the difference was statistically significant (P < 0.05, P < 0.01), as shown in Table 4.​ Figure 3 and Table 5.

[0119] Table 5 Comparison of GPX4 and Nrf2 protein expressions in kidney tissues of rats in each group by immunofluorescence detection

[0120]

[0121] Note: Compared with the sham operation group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P < 0.01.

[0122] In summary, the present invention successfully constructed a 5 / 6 nephrectomy-induced renal fibrosis model as a CKD model, which was characterized by abnormal renal function indicators, disordered renal structure, and significant tubular interstitial fibrosis in CKD rats. In this CKD model, iron levels rise due to the inability of intracellular iron to be excreted normally. The kidneys are under negative feedback regulation of high iron concentrations, TFR-1 protein expression is downregulated, and cells maintain homeostasis by increasing iron storage. Therefore, it also manifests as a renal fibrosis model caused by ferroptosis.

[0123] The present invention observes the effects of the Chinese medicine composition on CKD renal fibrosis involving ferroptosis from the following aspects.

[0124] (1) After intervention with the Chinese herbal composition of the present invention, the Scr, Bun, and 24h-Pro levels of CKD rats were significantly decreased, and the pathological damage to the kidneys of the rats was alleviated, indicating that the Chinese herbal composition has a protective effect on the kidneys of CKD rats.

[0125] (2) In the present invention, Fe in the kidney of CKD rats 2+ , MDA content increased significantly, GSH activity decreased significantly, and TFR-1 protein expression was also found to be downregulated. However, after intervention with the Chinese medicine composition of the present invention, Fe 2+ , MDA content was significantly reduced, GSH activity was significantly increased, and TFR-1 protein expression was significantly upregulated.

[0126] (3) Amino acid metabolism also plays an important role in ferroptosis. Inhibiting the activity of system Xc- (xCT) will hinder the absorption of cystine, thereby affecting the synthesis of GSH, and the antioxidant capacity of cells will be weakened. In this case, lipid reactive oxygen species continue to accumulate, eventually causing oxidative damage and ferroptosis of kidney cells. SLC7A11 is responsible for transporting extracellular amino acids into cells and reducing them to cysteine, which is the substrate for synthesizing GSH. GPX4 uses the reducing power provided by GSH to reduce phospholipid hydroperoxides to non-toxic alcohols, thereby preventing the further spread of lipid peroxidation. In CKD rats, the expression of SLC7A11, GPX4, and Nrf2 proteins is reduced, and GSH activity is reduced. Therefore, it is speculated that xCT may be inhibited, resulting in reduced GSH activity and decreased GPX4 protein expression, which weakens the tissue antioxidant capacity and ultimately induces ferroptosis. However, after intervention with the Chinese medicine composition of the present invention, the expression of SLC7A11, GPX4, and Nrf2 proteins in CKD rats was upregulated, and GSH activity was increased, which is consistent with the effect of losartan potassium. This shows that the Chinese medicine composition of the present invention can reduce kidney damage caused by ferroptosis by activating Nrf2 and upregulating the expression of SLC7A11 and GPX4, thereby playing a renal protective role in CKD rats.

[0127] (4) In ferroptosis, 4-HNE and GPX4 have a close interaction. The accumulation of 4-HNE will lead to the degradation or functional inactivation of GPX4, while the antioxidant function of GPX4 can inhibit the generation and toxicity of 4-HNE. In CKD rats, GPX4 protein expression was significantly reduced, and the content of 4-HNE, the end product of lipid peroxidation, increased, indicating that ferroptosis is involved in the progression of CKD renal fibrosis induced by 5 / 6 nephrectomy. However, after intervention with the Chinese medicine composition of the present invention, GPX4 protein expression in CKD rats was significantly increased, and the content of 4-HNE was significantly reduced, indicating that the Chinese medicine composition of the present invention can reduce the lipid peroxidation reaction in CKD rats and regulate ferroptosis.

[0128] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition, characterized in that It is composed of the following raw materials in parts by weight: 15-30 parts of Salvia miltiorrhiza, 9-12 parts of processed rhubarb, 8-18 parts of peach kernel, 5-15 parts of Chinese angelica, 9-15 parts of Cyathula, 4-10 parts of red peony root, 9-20 parts of Dendrobium officinale, 6-15 parts of Ligustrum lucidum fruit, 5-10 parts of Cornus officinalis, 9-15 parts of Centella asiatica, 3-6 parts of Panax notoginseng powder, and 10-30 parts of Astragalus.

2. The Chinese medicine composition according to claim 1, characterized in that It is composed of the following raw materials in parts by weight: 15 parts of Salvia miltiorrhiza, 9 parts of processed rhubarb, 12 parts of peach kernel, 12 parts of Chinese angelica, 15 parts of Cyathula, 9 parts of red peony root, 10 parts of Dendrobium officinale, 12 parts of Ligustrum lucidum fruit, 10 parts of Cornus officinalis, 10 parts of Centella asiatica, 6 parts of Panax notoginseng powder, and 30 parts of Astragalus.

3. Use of the traditional Chinese medicine composition according to any one of claims 1 or 2 in the preparation of a medicament for alleviating renal fibrosis caused by ferroptosis.

4. The use according to claim 3, characterized in that The medicine also includes pharmaceutical excipients.

5. The use according to claim 3, characterized in that The drug is a drug that increases the activity and / or expression of antioxidant-related proteins and / or small molecules.

6. The use according to claim 5, characterized in that The antioxidant-related protein is selected from any one or more of GPX4 and SLC7A11, and the small molecule substance is GSH.

7. The use according to claim 3, characterized in that The drug is a drug that increases the expression of proteins related to iron metabolism.

8. The use according to claim 7, characterized in that The protein related to iron metabolism is TFR-1.

9. The use according to claim 3, characterized in that The medicine is a medicine for reducing the content of lipid peroxidation end products.

10. The use according to claim 9, characterized in that The lipid peroxidation end product is selected from any one or more of 4-HNE and MDA.

Images