Application of wogonin in preparation of medicine for treating sjogren syndrome and traditional Chinese medicine composition for treating sjogren syndrome

Abstract

The invention provides application of wogonin in preparation of a medicine for treating sjogren syndrome and a traditional Chinese medicine composition for treating the sjogren syndrome, and belongs to the technical field of medicine preparation. The wogonin not only can inhibit the growth of M1 macrophages, but also can relieve the sjogren syndrome by inducing ferroptosis of the M1 macrophages. Meanwhile, the invention further provides a traditional Chinese medicine composition for treating the sjogren syndrome, and the composition contains wogonin and can be used for treating the sjogren syndrome.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to an application of wogonin in the preparation of a drug for treating Sjögren's syndrome and a traditional Chinese medicine composition for treating Sjögren's syndrome. Background Art

[0002] Wogonin is a flavonoid compound that shows potential anticancer activity in a variety of cancers. It can inhibit the occurrence and development of cancer by inhibiting cancer cell growth, invasion, migration and inducing ferroptosis. Studies have found that the combination of wogonin and some anti-tumor drugs (such as imatinib, sunitinib and cisplatin) can reverse the drug resistance of tumor treatment and ultimately improve the effect of tumor treatment. However, wogonin is less used in the treatment and application of some other autoimmune diseases.

[0003] Sjögren's syndrome Sjögren's syndrome (SS) is a chronic autoimmune disease that mainly invades the lacrimal glands and salivary glands, also known as autoimmune exocrine gland disease. It is mainly manifested as dry mouth, keratoconjunctivitis sicca, or other rheumatic diseases such as rheumatoid arthritis. It can involve other systems such as the respiratory system, digestive system, urinary system, blood system, nervous system, muscles, joints, etc., causing damage to multiple systems and organs. This disease can exist alone or in other autoimmune diseases. The one that exists alone is primary Sjögren's syndrome, while the one that is secondary to other autoimmune diseases such as rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus is secondary Sjögren's syndrome.

[0004] Ferroptosis is an iron-dependent programmed cell death. Its main mechanism is that under the action of ferrous iron or lipoxygenase, it catalyzes lipid peroxidation of unsaturated fatty acids highly expressed on the cell membrane, thereby inducing cell death. In addition, the phenomenon of ferroptosis is also manifested as a decrease in GPX4, the core enzyme regulating the antioxidant system (glutathione system), and significant changes in mitochondrial structure and morphology, mainly including mitochondrial shrinkage, increased mitochondrial membrane potential, and the disappearance of mitochondrial ridges. More and more evidence shows that ferroptosis plays an important role in regulating cell function and has theoretical guiding significance for the treatment of tumors and some autoimmune diseases.

[0005] Ferroptosis, as a new type of programmed cell death, is closely related to the occurrence and development of diseases. In recent years, traditional Chinese medicine has been widely used as a disease treatment drug. Its mechanism of action is relatively complex and has become a research hotspot. Therefore, studying the regulatory mechanism of wogonin on ferroptosis is crucial for its use in the preparation of drugs for the treatment of Sjögren's syndrome. Summary of the invention

[0006] In view of this, the present invention provides an application of wogonin in the preparation of a drug for treating Sjögren's syndrome. Wogonin can not only inhibit the growth of M1 macrophages, but also alleviate Sjögren's syndrome by inducing ferroptosis of M1 macrophages.

[0007] The purpose of the present invention is achieved through the following solutions:

[0008] A use of wogonin in preparing a medicine for treating Sjögren's syndrome.

[0009] Preferably, the medicine is a traditional Chinese medicine composition comprising wogonin.

[0010] A traditional Chinese medicine composition for treating Sjögren's syndrome comprises the following raw materials in parts by weight: 10-20 parts of wogonin, 1-5 parts of luteolin, 1-5 parts of quercetin, 1-5 parts of isoliquiritigenin, 1-5 parts of hesperidin, 1-5 parts of curcuminol, 1-5 parts of squalene, 0-5 parts of fisetin, 0-5 parts of polydatin, and 0-5 parts of proanthocyanidins.

[0011] The method for preparing the Chinese medicine composition for treating Sjögren's syndrome comprises the following steps:

[0012] (1) weighing raw materials according to proportion, and crushing the raw materials in a crusher to a particle size of less than 100 mesh, and then mixing them thoroughly to obtain a mixture;

[0013] (2) Add 2 to 3 times the weight of water to the mixture described in step (1), dissolve it at 60 to 70° C. under ultrasound for 30 to 40 minutes, collect the filtrate after filtering, spray dry to obtain a solid powder, prepare a granular preparation, and obtain the Chinese medicine composition for treating Sjögren's syndrome.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides an application of wogonin in the preparation of a drug for treating Sjögren's syndrome. Wogonin can not only inhibit the growth of M1 macrophages, but also relieve Sjögren's syndrome by inducing ferroptosis of M1 macrophages. At the same time, the present application provides a Chinese medicine composition for treating Sjögren's syndrome, which contains wogonin and can be used to treat Sjögren's syndrome. Mouse experiments have shown that the Chinese medicine combination can promote the ferroptosis of M1 macrophages, and the symptoms of Sjögren's syndrome are alleviated. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Flow cytometry detection of macrophages in clinical samples of Sjögren's syndrome patients;

[0017] Figure 2Flow cytometry detection of macrophages in a mouse model of Sjögren's syndrome;

[0018] Figure 3 Flow cytometry was used to detect the induced M1 macrophages;

[0019] Figure 4 The activity level of CCK8 after WOG treatment in M1 macrophages was detected;

[0020] Figure 5 IC50 value determination for WOG-treated M1 macrophages;

[0021] Figure 6 To detect the apoptosis level of M1 macrophages after WOG treatment;

[0022] Figure 7 To detect the activity levels of M1 macrophages after treating them with various death inhibitors combined with WOG;

[0023] Figure 8 Detection of ferroptosis metabolites after WOG treatment of M1 macrophages;

[0024] Fig. 9 mRNA detection of ferroptosis-related markers in M1 macrophages before and after WOG treatment;

[0025] Fig.10 The protein levels of ferroptosis-related markers were detected in M1 macrophages before and after WOG treatment;

[0026] Fig.11 Flow cytometry was used to detect the level of ROS in M1 macrophages after treatment with WOG;

[0027] Fig.12 It is a bar graph analysis of ROS levels in M1 macrophages after WOG treatment;

[0028] Fig.13 Flow cytometry was used to detect the lipid peroxidation level of M1 macrophages after WOG treatment;

[0029] Fig.14 It is a bar graph analysis of lipid peroxidation levels in M1 macrophages after WOG treatment;

[0030] Fig.15 To detect the protein levels of FOXM1 and p-FOXM1 in M1 macrophages after WOG treatment;

[0031] Fig.16 To detect the mRNA level of FOXM1 in M1 macrophages after WOG treatment;

[0032] Fig.17To verify that FOXM1 regulates GPX4 gene transcription for CUT&RUN experiments;

[0033] Fig.18 To detect the distribution of FOXM1 in the nucleus and cytoplasm after WOG treatment of M1 macrophages;

[0034] Fig.19 Flow cytometry was used to detect the level of ROS in M1 macrophages in ESS model mice after treatment with traditional Chinese medicine;

[0035] Fig. 20 The HE experiment was used to detect the pathological status of salivary gland tissue in ESS model mice after treatment with traditional Chinese medicine. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the embodiments.

[0037] Example 1

[0038] M1 macrophages were treated with different concentrations of wogonin (WOG, MCE, HY-N0400) as follows:

[0039] Step 1. Detection of macrophages in clinical samples

[0040] Peripheral blood was collected from patients with Sjögren's syndrome (SS) and healthy controls (HC), and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll reagent (MCE, HY-111782) and density gradient centrifugation. The PBMCs isolated were then incubated with flow cytometry antibodies FITC-CD86 (Invitrogen, MA1-10300) and APC-CD163 (Invitrogen, MA5-17717), and the changes in the proportion of M1 and M2 macrophages were detected by flow cytometry. The results are shown in Figure 2. Figure 1 .

[0041] Step 2: Detection of macrophages in the Sjögren's syndrome mouse model

[0042] 2.1 Construction of mouse model

[0043] Bilateral salivary gland proteins were isolated from female C57BL / 6 mice (8 weeks old), emulsified to 2 mg / mL in an equal volume of Freund's complete adjuvant CFA (MCE, HY-153808B), and injected subcutaneously into naturally immunized mice (6-week-old male mice) in the neck on days 0 and 7, respectively. On day 14, salivary gland proteins emulsified with Freund's incomplete adjuvant IFA (MCE, HY-153808A) were boosted.

[0044] 2.2 Detection of macrophages in mouse models

[0045] For the above-mentioned Sjögren's syndrome model mice, some lesions were removed, and an equal number of healthy mice were selected to take samples from the same parts. Single cells were prepared using a tissue single cell preparation kit, and then flow cytometry antibodies were used to detect the difference in the proportion of M1 and M2 macrophages. The results are as follows: Figure 2 .

[0046] Step 3: Induction of THP1 differentiation into M1 macrophages in vitro

[0047] The cells selected for this part of the experiment are THP1 cells. Different cytokine stimulation can be added in vitro to induce differentiation into M1 and M2. The processing process is as follows:

[0048] 3.1 THP1 cell culture

[0049] (1) Complete medium: 1640 (MCE, HY-153808A) containing 10% FBS and 0.05 mM β-mercaptoethanol (MCE, HY-Y0326). The culture conditions were 37°C and 5% CO2.

[0050] (2) Cell culture: When the cell density reaches 80%-90%, the cells are subcultured and placed back into a 37°C, 5% CO2 cell culture incubator for further culture.

[0051] 3.2 Induction and differentiation of M1 cells and their identification

[0052] THP1 cells were evenly plated in a 6-well plate, and PMA (phorbol-12-myristic acid-13-acetate) (MCE, HY-18739) was added to an appropriate concentration and cultured for 48 hours to induce cell differentiation into M0 macrophages. PMA was removed, and IFN-γ (interferon-γ) (MCE, HY-P4717A) and LPS (lipopolysaccharide) (MCE, HY-D1056) were added to an appropriate concentration and cultured for 48 hours to induce M1 polarization. Flow cytometry was performed using FITC-CD86 flow cytometry antibody, and the results were shown in Figure 2. Figure 3 .

[0053] Step 4: Treat M1 macrophages with different concentrations of WOG

[0054] The processing process is as follows:

[0055] (1) M1 macrophages were evenly plated in a 6-well plate. When the density reached about 70%, WOG was added in sequence to concentrations of 0, 5, 10, 20, 40, and 80 μM, respectively, and treated for 24 h.

[0056] (2) Protein and mRNA were collected separately to detect the gene transcription level and protein expression level of ferroptosis-related molecules.

[0057] Step 5: Detection of cell activity level after WOG treatment of M1 macrophages

[0058] The CCK-8 kit was used to detect the changes in cell activity of M1 macrophages before and after treatment with different concentrations of WOG (concentrations were 0, 5, 10, 20, 40 and 80 μM). After 0 h, 24 h, 36 h, 48 h and 72 h, the effect of WOG at different concentrations on the activity of M1 macrophages was detected by an ELISA instrument. The results are as follows Figure 4 and Figure 5 .

[0059] Step 6. Detection of apoptosis level in M1 macrophages after WOG treatment

[0060] After M1 macrophages were treated with different concentrations of WOG, the level of cell apoptosis was detected by flow cytometry

[0061] After M1 macrophages were treated with different concentrations of WOG (0, 5, 10, 20, 40 and 80 μM) for 24 h, the apoptosis level of M1 macrophages in each treatment group was determined by flow cytometry. Figure 6 .

[0062] Step 7: Combined treatment of M1 macrophages with inhibitors of multiple death pathways and WOG

[0063] Different types of cell death inhibitors were used to treat M1 macrophages in combination with WOG. The cell death inhibitors mainly included: apoptosis inhibitor Z-VAD-FMK (Z-VAD) (MCE, HY-16658B), autophagy inhibitor 3-methyladenine (3-ME) (MCE, HY-19312), pyroptosis inhibitor Bay 11-7821 (BAY) (MCE, HY-13453), necroptosis inhibitor Necrosulfonamide (NSA) (MCE, HY-100573) and ferroptosis inhibitor (Fer-1) (MCE, HY-100579). The experimental groups in this part were set up as follows: CTRL (no drug treatment group), DMSO (MCE, HY-Y0320) treatment group (negative control group), WOG treatment group, WOG combined with Fer-1 treatment group, WOG combined with Z-VAD treatment group, WOG combined with 3-ME treatment group, WOG combined with BAY treatment group, and WOG combined with NSA treatment group. After 48 h of combined treatment, the cell activity in each sample was detected by CCK8. The results are shown in Figure 7 By analyzing the changes in cell activity after combined treatment with each inhibitor and WOG, it is possible to determine the mode of death through which WOG inhibits the occurrence and development of tumors.

[0064] Step 8. Detection of ferroptotic metabolites before and after WOG treatment

[0065] Detection of ferroptosis metabolites in M1 macrophages after treatment with different concentrations of WOG

[0066] M1 macrophages were treated with different concentrations of WOG and combined with ferroptosis inhibitors Fer-1 and Lip-1 (MCE, HY-P74352). MDA (Beyotime, S0131S) and GSH (Beyotime, S0052) detection kits were used to detect the levels of ferroptosis-related metabolites MDA and GSH. The results are shown in Figure 2. Figure 8 .

[0067] Step 9. Detection of mRNA and protein levels of ferroptosis-related markers before and after WOG treatment

[0068] 9.1. Detection of mRNA of ferroptosis-related markers after treatment of M1 macrophages with different concentrations of WOG

[0069] M1 macrophages were treated with different concentrations of WOG for 24 h and then the cells were collected (the treatment concentrations were 0, 5, 10, 20, 40 and 80 μM, respectively). Total RNA was extracted using Trizol (Vazyme, R401-01) reagent and reverse transcribed into cDNA using HiScriptIIQ RTSuperMix (Vazyme, R222-01) for qPCR (AceQ qPCR SYBR GreenMasterMix) (Vazyme, Q111-02 / 03). The genes detected mainly include: GPX4, SLC7A11, ACSL4, ACSL3, FTH1 and FSP1. The results are shown in Figure 2. Fig. 9 .

[0070] 9.2 Protein detection of ferroptosis-related markers after treatment of M1 macrophages with different concentrations of WOG

[0071] M1 macrophages were treated with different concentrations of WOG for 48 h and then the cells were collected (the treatment concentrations were 0, 5, 10, 20, 40 and 80 μM, respectively), and total protein was extracted using RIPA (Ncmbio, WB3100) reagent. The protein molecules detected mainly included: GPX4 (Proteintech, 30388-1-AP), SLC7A11 (Proteintech, 26864-1-AP), ACSL4 (Proteintech, 22401-1-AP), ACSL3 (Proteintech, 20710-1-AP), FTH1 (Proteintech, 11682-1-AP) and FSP1 (Proteintech, 20886-1-AP). The results are shown in Figure 2. Fig.10 .

[0072] Step 10: Detection of ROS and lipid peroxidation levels in M1 macrophages before and after treatment by flow cytometry

[0073] 10.1. Detection of ROS levels by flow cytometry after treatment of M1 macrophages with different concentrations of WOG

[0074] M1 macrophages were treated with different concentrations of WOG for 24 h and then the cells were collected (the treatment concentrations were 0, 5, 10, 20, 40 and 80 μM, respectively). The H2DCFDA detection probe (MCE, HY-D0940) was used to detect the intracellular ROS level in each group of samples. The results are shown in Figure 2. Fig.11 and Fig.12 .

[0075] 10.2. Flow cytometry was used to detect lipid peroxidation levels in M1 macrophages after treatment with different concentrations of WOG

[0076] M1 macrophages were treated with different concentrations of WOG for 24 hours and then the cells were collected (the treatment concentrations were 0, 5, 10, 20, 40 and 80 μM). The level of intracellular lipid peroxidation in each group of samples was detected using C11-BODIPY detection probe (MCE, HY-D1301). The results are shown in Figure 2. Fig.13 and Fig.14 .

[0077] Step 11. Detection of FOXM1 gene transcription and protein expression levels before and after WOG treatment of M1 macrophages

[0078] 11.1. Detection of FOXM1 mRNA in M1 macrophages after treatment with different concentrations of WOG

[0079] M1 macrophages were treated with different concentrations of WOG for 24 h and then the cells were collected (the treatment concentrations were 0, 5, 10, 20, 40 and 80 μM, respectively). Total RNA was extracted using Trizol reagent and reverse transcribed into cDNA using HiScript II Q RT SuperMix, and used for qPCR (AceQ qPCR SYBR Green Master Mix) to detect the mRNA level of FOXM1. The results are shown in Fig.16 .

[0080] 11.2. Protein detection of FOXM1 and p-FOXM1 in M1 macrophages after treatment with different concentrations of WOG

[0081] M1 macrophages were treated with different concentrations of WOG for 48 h, and then the cells were collected (the treatment concentrations were 0, 5, 10, 20, 40 and 80 μM, respectively). Total protein was extracted with RIPA reagent, and the protein levels of FOXM1 (Proteintech, 13147-1-AP) and p-FOXM1 (CST, 14170S) were detected by Western Blot. The results are shown in Fig.15 .

[0082] Step 12: CUT&RUN experiments verify that FOXM1 regulates GPX4 gene transcription

[0083] This part of the experiment used the HyperactivepG-MNase CUT&RUNAssayKit for PCR / qPCR (Vazyme, HD101-01 / 02) kit to verify whether FOXM1 regulates the gene transcription of GPX4 in M1 macrophages. The experimental process is as follows: collect cells, incubate FOXM1 primary antibody, MNase bind, gene fragment and terminate, release and extract DNA, and finally perform RTPCR detection. The multiple pairs of quantitative primers used in this part are designed according to the promoter region of the GPX4 gene. At the same time, in the experiment, the present invention designed an experimental negative control group, namely the IgG treatment group. The results are as follows. Fig.17 .

[0084] Step 13: Nuclear and cytoplasmic separation of transcription factor FOXM1

[0085] After M1 macrophages were treated with different concentrations of WOG for 24 hours, the cells were collected (the treatment concentrations were 0 and 40 μM, respectively), and 100 μL of hypotonic buffer was added, resuspended, and placed on ice for 15 minutes. 0.2% TritonX-100 (Beyotime, P0096-500ml) was added, and high-intensity vortexing was performed for 10 seconds. Centrifugation was performed at 3500 rpm for 10 minutes at 4°C. At this time, the supernatant was the cytoplasm and the precipitate was the nucleus. The supernatant and the precipitate were transferred to new centrifuge tubes, and protein loading 5× Loading (Ncmbio, WB3002) was added. After boiling at 100°C for 10 minutes, the samples were tested by Western Blot test. The results are shown in Figure 2. Fig.18 .

[0086] First, multiple pairs of clinical samples were selected, mainly including peripheral blood of healthy (HC) and Sjögren's syndrome patients (SS), and the differences in macrophages between the healthy group and the Sjögren's syndrome group were detected by flow cytometry, mainly detecting the typing and proportion of macrophages. The results showed that in Sjögren's syndrome patients, M1 macrophages were significantly increased and M2 cells were significantly downregulated ( Figure 1 ). Subsequently, the present invention further explored the Sjögren's syndrome mouse model by detecting the macrophages infiltrating the tissues of the lesion sites of the Sjögren's syndrome model mice. The results showed that in the Sjögren's syndrome model mice, the proportion of M1 macrophages was much higher than that of normal mice, while the proportion of M2 macrophages was the opposite ( Figure 2 ). Through the detection of Sjögren's syndrome clinical samples and mouse models, it is shown that M1 macrophages play an important role in the disease of Sjögren's syndrome, and aggravate the symptoms of Sjögren's syndrome through its pro-inflammatory mechanism. Therefore, the later stage of the present invention will further study the regulatory mechanism of wogonin on M1 macrophages, which provides a basis for the treatment of Sjögren's syndrome with wogonin.

[0087] In order to explore the mechanism of wogonin (WOG) inducing ferroptosis in M1 macrophages, the present invention first constructed an induction model of M1 macrophages in vitro. After directional induction, the M1 macrophages ( Figure 3 ). Then, different concentrations of wogonin (including 0, 5, 10, 20, 40, 80 μM) were used to treat M1 macrophages to detect their effects on activity. After WOG treatment, it was found that the proliferation ability of M1 macrophages was significantly inhibited, and this inhibitory effect showed dose- and time-dependent effects ( Figure 4 ). Furthermore, the IC50 value of WOG-treated M1 macrophages was 98.2 μM ( Figure 5 In the experiment, the present invention further found that WOG can induce apoptosis of tumor cells in a concentration-dependent manner using flow cytometry ( Figure 6 ). In order to further confirm that WOG can induce ferroptosis in tumor cells, the present invention uses inhibitors of multiple cell death modes combined with WOG to treat M1 macrophages. Through CCK8 experiments, the results show that the cell activity of M1 macrophages is most significantly reduced when treated with 80μM concentration of WOG. Further treatment with ferroptosis inhibitor (Fer-1) combined with WOG found that this inhibitory activity can be effectively reversed, and the obtained cell activity level is equivalent to that of the control group, while inhibitors of other cell death pathways cannot or can only partially restore cell viability ( Figure 7 ).

[0088] In order to further confirm that WOG can significantly inhibit the cell activity of M1 macrophages through ferroptosis, the present invention further detected the intracellular levels of ferroptosis-related markers, such as MDA and GSH. The results showed that WOG can significantly increase the intracellular level of MDA, a metabolite of ferroptosis, while downregulating the level of GSH, an intracellular antioxidant that inhibits ferroptosis. Interestingly, the changes in the levels of these ferroptosis-related markers can be reversed by ferroptosis inhibitors (Fer-1 or Lip-1) ( Figure 8 ). Therefore, these results indicate that WOG can significantly inhibit the occurrence and development of Sjögren's syndrome by inducing ferroptosis of M1 macrophages, further expounding the regulatory mechanism of wogonin on macrophage ferroptosis, and laying a theoretical foundation for it as a new macrophage ferroptosis inducer. In addition, the changes in the mRNA levels of genes related to ferroptosis were verified by RT-PCR experiments. The results showed that the gene transcription level of GPX4 decreased significantly with WOG treatment in a dose-dependent manner, while the mRNA levels of other molecules (ACSL4, ACSL3, SLC7A11, FSP1 and FTH1) were not affected by WOG ( Fig. 9 ). Subsequently, the protein levels of the above molecules were detected by Western Blot experiments, and similar experimental results were obtained as before, that is, WOG can cause a significant downregulation of GPX4 protein, while having no effect on the protein levels of other molecules. These experimental results indicate that WOG may induce ferroptosis in M1 macrophages by downregulating the level of GPX4 ( Fig.10 ). Subsequently, flow cytometry was used to detect the changes in intracellular ROS and lipid peroxidation levels in M1 macrophages after WOG treatment. The results showed that compared with the control group (WOG 0μM), WOG treatment could induce M1 macrophages to produce high levels of ROS and lipid peroxidation, which was most obvious at WOG 80μM concentration, which further indicated that WOG could act as an inducer of ferroptosis to induce ferroptosis in M1 macrophages ( Fig.11 , Fig.12 , Fig.13 and Fig.14 ). Through a large number of literature readings, it was found that FOXM1 can act as a transcription factor to regulate the gene transcription of GPX4. Therefore, the protein expression of FOXM1 in M1 macrophages after WOG treatment was subsequently detected by Western Blot. The results showed that with the treatment of WOG, the protein level of FOXM1 was significantly inhibited ( Fig.15 As an important gene transcription factor, FOXM1 can only play its gene transcription function when it is phosphorylated and enters the nucleus. Therefore, the present invention subsequently detected the phosphorylation level of FOXM1 (p-FOXM1). The results showed that the expression trend of p-FOXM1 was similar to that of FOXM1 protein, and both showed a decrease in a WOG concentration-dependent manner ( Fig.15 ). At the same time, the present invention also detected the changes in the mRNA level of FOXM1, and found that the mRNA level of FOXM1 was also significantly inhibited by WOG ( Fig.16 ). Subsequently, through CUT&RUN experiments, it was found that the transcription factor FOXM1 can bind to the -1200 to -1000 sites upstream of the GPX4 gene promoter, thus confirming that FOXM1 can regulate the gene transcription of GPX4 ( Fig.17 ). Finally, by Western Blot experiment, FOXM1 and p-FOXM1 in the cytoplasm and nucleus were detected respectively. The results showed that in M1 macrophages treated with WOG, both FOXM1 and p-FOXM1 were significantly downregulated, especially the protein level of p-FOXM1. The results further showed that with the treatment of WOG, the phosphorylation level of FOXM1 was significantly weakened, and the present invention also found that with the treatment of WOG, the nuclear entry of p-FOXM1 was sharply reduced, thereby inhibiting the gene transcription of downstream GPX4, and finally the GPX4 protein level was reduced, thereby promoting the ferroptosis of M1 macrophages ( Fig.18 ).

[0089] Example 2

[0090] A traditional Chinese medicine composition for treating Sjögren's syndrome is composed of the following raw materials in parts by weight: 15 parts of wogonin, 2 parts of luteolin, 1 part of quercetin, 1 part of isoliquiritigenin, 1 part of hesperidin, 1 part of curcuminol, 2 parts of squalene and 1 part of fisetin.

[0091] The preparation method of the Chinese medicine composition for treating Sjögren's syndrome comprises the following steps:

[0092] (1) weighing raw materials according to proportion, and crushing the raw materials in a crusher to a particle size of less than 100 mesh, and then mixing them thoroughly to obtain a mixture;

[0093] (2) Add 2 times the weight of water to the mixture described in step (1), dissolve it at 70° C. for 30 minutes under ultrasound, collect the filtrate after filtering, spray-dry to obtain a solid powder, prepare a granular preparation, and obtain the Chinese medicine composition for treating Sjögren's syndrome.

[0094] Example 3

[0095] A Chinese medicine composition for treating Sjögren's syndrome, comprising the following raw materials in parts by weight:

[0096] 20 parts of wogonin, 2 parts of luteolin, 1 part of quercetin, 1 part of isoliquiritigenin, 1 part of hesperidin, 1 part of curcuminol, 2 parts of squalene and 1 part of fisetin.

[0097] The preparation method of the Chinese medicine composition is the same as that of Example 2.

[0098] Example 4

[0099] A Chinese medicine composition for treating Sjögren's syndrome, comprising the following raw materials in parts by weight:

[0100] 15 parts of wogonin, 1 part of luteolin, 1 part of quercetin, 1 part of isoliquiritigenin, 1 part of hesperidin, 1 part of curcuminol, 2 parts of squalene and 1 part of polydatin.

[0101] The preparation method of the Chinese medicine composition is the same as that of Example 2.

[0102] Example 5

[0103] A Chinese medicine composition for treating Sjögren's syndrome, comprising the following raw materials in parts by weight:

[0104] 15 parts of wogonin, 1 part of luteolin, 1 part of quercetin, 1 part of isoliquiritigenin, 1 part of hesperidin, 1 part of curcuminol, 2 parts of squalene, 1 part of fisetin, and 1 part of naringenin.

[0105] The preparation method of the Chinese medicine composition is the same as that of Example 2.

[0106] Example 6

[0107] A Chinese medicine composition for treating Sjögren's syndrome, comprising the following raw materials in parts by weight:

[0108] 15 parts of wogonin, 1 part of luteolin, 1 part of quercetin, 1 part of isoliquiritigenin, 1 part of hesperidin, 1 part of curcuminol, 2 parts of squalene, 1 part of fisetin, 1 part of naringenin, and 1 part of proanthocyanidins.

[0109] The preparation method of the Chinese medicine composition is the same as that of Example 2.

[0110] The Chinese medicine composition for treating Sjögren's syndrome of Example 2 and Example 6 was used to treat Sjögren's syndrome mice, and the steps were as follows:

[0111] The above-mentioned successfully constructed Sjögren's syndrome model mice were selected and randomly divided into 6 groups, 4 groups in each group, and treated with oral gavage respectively, and the dosage was 30 mg / kg. The specific treatment groups are as follows: the first group used an equal volume of normal saline as the control group; the second group was the drug treatment group of Example 2 (W1); the third group was the drug treatment group of Example 3 (W2); the fourth group was the drug treatment group of Example 4 (W3); the fifth group was the drug treatment group of Example 5 (W4); and the sixth group was the drug treatment group of Example 6 (W5). After continuous oral gavage treatment for 10 days, the lesion site (salivary gland) of each experimental group was taken out after the experiment. A part of the tissue was used to prepare single cells using a tissue single cell preparation kit, and then the level of ROS in the M1 macrophages of each mouse was detected by flow cytometry. The results are shown in the following figure. Fig.19 The other part of the tissue was used for HE staining experiments to further verify the therapeutic effect of the Chinese medicine combination. The results are as follows Fig. 20 .

[0112] The results of flow cytometry showed that compared with the control group, the level of ROS in M1 macrophages in ESS model mice treated with Chinese herbal combination drugs (W1, W2, W3, W4, W5) was significantly upregulated, indicating that Chinese herbal combination drugs can promote the occurrence of ferroptosis in M1 macrophages ( Fig.19 HE staining experiments showed that the pathology of the lesion sites of ESS model mice treated with the Chinese herbal medicine combination (W1, W2, W3, W4, W5) was significantly restored compared with the control group. For example, the lymphocyte infiltration in the salivary gland tissue of the mice was reduced and the damaged gland ducts were significantly restored, that is, the symptoms of Sjögren's syndrome were alleviated ( Fig. 20 ).

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of wogonin in the preparation of a medicine for treating Sjögren's syndrome.

2. The use of wogonin according to claim 1 in the preparation of a medicament for treating Sjögren's syndrome, characterized in that: The medicine is a traditional Chinese medicine composition containing wogonin.

3. A Chinese medicine composition for treating Sjögren's syndrome, characterized in that: The invention comprises the following raw materials in parts by weight: 10-20 parts of wogonin, 1-5 parts of luteolin, 1-5 parts of quercetin, 1-5 parts of isoliquiritigenin, 1-5 parts of hesperidin, 1-5 parts of curcuminol, 1-5 parts of squalene, 0-5 parts of fisetin, 0-5 parts of polydatin and 0-5 parts of proanthocyanidins.

4. The method for preparing the Chinese medicine composition for treating Sjögren's syndrome according to claim 3, characterized in that: The following steps are involved: (1) weighing raw materials according to proportion, and crushing the raw materials in a crusher to a particle size of less than 100 mesh, and then mixing them thoroughly to obtain a mixture; (2) Add 2 to 3 times the weight of water to the mixture described in step (1), dissolve it at 60 to 70° C. under ultrasound for 30 to 40 minutes, collect the filtrate after filtering, spray dry to obtain a solid powder, prepare a granular preparation, and obtain the Chinese medicine composition for treating Sjögren's syndrome.

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