Use of reagent for detecting expression level of gstm3 in preparation of product for predicting or diagnosing liver fibrosis reversal

Abstract

The application provides a reagent for detecting the expression level of GSTM3 in the preparation of a product for predicting or diagnosing liver fibrosis reversal, and belongs to the technical field of disease detection reagents and therapeutic drugs. The application research finds that, compared with liver fibrosis mice, the expression level of GSTM3 in liver fibrosis reversal mice is significantly decreased, indicating that the expression level of GSTM3 can be accurately predicted or diagnosed for liver fibrosis reversal. In addition, compared with activated hepatic stellate cells, after the expression of GSTM3 in the activated hepatic stellate cells is knocked down, the expression levels of liver fibrosis related proteins alpha-SMA, COL1A1, Elastin and Fibronectin are significantly reduced, indicating that a GSTM3 inhibitor can be used for treating liver fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of disease detection reagents and therapeutic drugs, and particularly relates to application of a reagent for detecting expression level of GSTM3 in preparation of a product for predicting or diagnosing liver fibrosis reversal. BACKGROUND

[0002] Liver fibrosis is a result of chronic liver damage and repair imbalance caused by viral infection, metabolic disorder, immune abnormality and other pathogenic factors, and is also a necessary stage for various chronic liver diseases to transform into cirrhosis, liver failure and even liver cancer. According to statistics, the number of people who die of liver diseases worldwide each year exceeds 2 million, accounting for about 4% of the total number of deaths in the world (Devarbhavi H, Asrani SK, Arab JP, Na Realtimeey YA, Pose E, Kamath PS. Global burden of liver disease: 2023 update. J Hepatol. 2023 Aug; 79(2): 516-537. doi: 10.1016 / j.jhep.2023.03.017. Epub 2023 Mar 27. PMID: 36990226). Among the liver cancer patients who rank fourth in the number of cancer deaths worldwide, 80% to 90% develop from hepatitis, liver fibrosis and cirrhosis (Li Caojie, Li Jiajun, Chen Rongxin. Research Progress of Liver Fibrosis and Immune Microenvironment of Liver Cancer [J]. Fudan University Journal (Medical Science), 2023, 50(06): 889-896). For a long time, liver fibrosis has been considered as an irreversible pathological process. However, with the rapid development of omics technology and the in-depth study of the liver, more and more research data show that liver fibrosis can be reversed to varying degrees if the pathogenic factors of chronic liver damage can be effectively eliminated. At present, it is believed that the core mechanism of liver fibrosis reversal mainly depends on the phenotypic transformation of hepatic stellate cells (from activated state to resting state), extracellular matrix degradation mediated by matrix metalloproteinases, and regeneration and repair of liver parenchymal cells. However, the existing clinical evaluation system has obvious limitations. Although liver biopsy is the gold standard, it is invasive, and the sensitivity and specificity of non-invasive diagnostic indicators (such as serological markers and transient elastography) in dynamic monitoring of fibrosis reversal still need large-scale clinical verification. Therefore, systematic analysis of the molecular mechanism of liver fibrosis reversal and finding diagnostic markers and therapeutic targets with clinical transformation value are of important theoretical significance and practical value for promoting precise diagnosis and treatment of liver fibrosis.

[0003] Glutathione S-transferase (GST) is a phase II enzyme system for detoxification of exogenous compounds, and its core function is to catalyze the combination of glutathione and various electrophilic compounds, thereby participating in the detoxification of carcinogens and the metabolic regulation of various bioactive compounds. As one of the members of this family, GSTM3 gene plays an important role in the detoxification of electronic compounds (such as toxins, carcinogens) and the regulation of oxidative stress. Whether GSTM3 gene is involved in the process of liver fibrosis reversal and its specific mechanism of action is still a scientific problem that has not been clearly defined. SUMMARY

[0004] In view of this, the purpose of the present application is to provide a reagent for detecting the expression level of GSTM3 in the preparation of a product for predicting or diagnosing liver fibrosis reversal.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides a reagent for detecting the expression level of GSTM3 in the preparation of a product for predicting or diagnosing liver fibrosis reversal.

[0007] Preferably, the reagent for detecting the expression level of GSTM3 includes a GSTM3 antibody or a primer pair for detecting the expression of GSTM3.

[0008] Preferably, the sequence of the primer pair for detecting the expression of GSTM3 is shown in SEQ ID NO. 3-SEQ ID NO. 4.

[0009] The present application provides a reagent for detecting the expression level of GSTM3 in the preparation of a product for predicting or diagnosing liver fibrosis or liver fibrosis progression grading.

[0010] The present application provides a use of a GSTM3 inhibitor in the preparation of a product for treating liver fibrosis.

[0011] Preferably, the GSTM3 inhibitor includes one or both of a reagent for reducing the expression of GSTM3 and a modulator for reducing the product of GSTM3.

[0012] Preferably, the reagent for reducing the expression of GSTM3 includes a reagent for knocking down or silencing GSTM3; and the modulator for reducing the product of GSTM3 includes a GSTM3 antibody or a protease for degrading the product of GSTM3.

[0013] Preferably, the reagent for knocking down or silencing GSTM3 includes siRNA, shRNA or miRNA.

[0014] Preferably, the siRNA consists of a sense strand and an antisense strand, the sequence of the sense strand is shown as SEQ ID NO. 1, and the sequence of the antisense strand is shown as SEQ ID NO. 2.

[0015] The present application provides a drug for treating liver fibrosis, the drug comprising siRNA, the siRNA consisting of a sense strand and an antisense strand, the sequence of the sense strand is shown as SEQ ID NO. 1, and the sequence of the antisense strand is shown as SEQ ID NO. 2.

[0016] The present application provides a system for predicting or diagnosing liver fibrosis reversal, comprising a data processing device and a detection device, the detection device being used for detecting the expression level of GSTM3 in a sample to be tested and a sample of a liver fibrosis patient; the data processing device comprises a conclusion output module, if the expression level of GSTM3 in the sample to be tested is significantly reduced compared with the expression level of GSTM3 in the sample of the liver fibrosis patient, the sample to be tested is predicted or diagnosed to have fibrosis reversal; the sample to be tested is a liver fibrosis patient who is receiving drug treatment.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application provides a use of a reagent for detecting the expression level of GSTM3 in the preparation of a product for predicting or diagnosing liver fibrosis reversal, the present application researches and finds that, compared with liver fibrosis mice, the expression level of GSTM3 in liver fibrosis reversal mice is significantly reduced, indicating that the expression level of GSTM3 can be accurately predicted or diagnosed to have liver fibrosis reversal by detecting the expression level of GSTM3. In addition, compared with activated hepatic stellate cells, after the expression of GSTM3 in the activated hepatic stellate cells is knocked down, the expression levels of liver fibrosis related proteins α-SMA, COL1A1, Elastin and Fibronectin are significantly reduced, indicating that the GSTM3 inhibitor can be used for treating liver fibrosis. GSTM3 provides a new target for treating or diagnosing liver fibrosis and predicting or diagnosing liver fibrosis reversal. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 For the liver fibrosis reversal mouse model and proteomics analysis results, A: heat map of differential proteins in serum and liver tissue samples of liver fibrosis reversal model mice; B: H&E, Masson, GSTM3 immunohistochemical staining results of liver tissue of liver fibrosis reversal model mice, the column chart in B is the statistical result of the expression level of GSTM3 detected by immunohistochemical staining, and the column chart on the right is the statistical result of the collagen fiber area of each group after Masson staining; C: Western blot detection of GSTM3 protein level in liver tissue of liver fibrosis and reversal model mice;

[0020] Figure 2 To detect the content of GSTM3 in the plasma of healthy people and liver fibrosis patients by using the ELISA kit respectively;

[0021] Figure 3 To study the influence of GSTM3 knockout on the quiescent and activated hepatic stellate cells, A: Realtime PCR was used to detect the mRNA level of GSTM3 in the GSTM3 knockout cells; B: Western blot was used to detect the protein expression of GSTM3 in the GSTM3 knockout cells; C: Western blot was used to detect the protein level of fibrosis markers in the GSTM3 knockout cells; D: Realtime PCR was used to detect the mRNA level of α-SMA, COL1A1 and TIMP1 in the LX-2 cells and the GSTM3 knockout cells after TGF-β stimulation; E: Western blot was used to detect the protein level of fibrosis markers in the LX-2 cells after TGF-β stimulation and GSTM3 knockout;

[0022] Figure 4 To study the influence of GSTM3 overexpression on the hepatic stellate cells, A: Western blot was used to detect the protein expression level of GSTM3 in the LX-2 cells with GSTM3 overexpression; B: Western blot was used to detect the protein level of fibrosis markers in the LX-2 cells with GSTM3 overexpression. DETAILED DESCRIPTION

[0023] The application provides a reagent for detecting the expression level of GSTM3 in the preparation of a product for predicting or diagnosing the reversal of liver fibrosis.

[0024] In the application, the reagent for detecting the expression level of GSTM3 comprises a GSTM3 antibody or a primer pair for detecting the expression of GSTM3. The method for detecting the expression of GSTM3 can adopt the method of Western Blot or Realtime PCR. The sequence of the primer pair for detecting the expression of GSTM3 is shown in SEQ ID NO. 3-SEQ ID NO. 4. The application finds that the expression level of GSTM3 in the liver fibrosis reversal mice is significantly reduced compared with the liver fibrosis model mice, and therefore, the GSTM3 can be used as a detection target for predicting or diagnosing the reversal of liver fibrosis.

[0025] The application provides application of a reagent for detecting expression level of GSTM3 in preparation of a product for predicting or diagnosing liver fibrosis or grading progression of liver fibrosis.

[0026] The application provides application of a GSTM3 inhibitor in preparation of a product for treating liver fibrosis.

[0027] In the application, it is found that, by knocking down the expression level of GSTM3 in activated hepatic stellate cells, the activation of the hepatic stellate cells can be effectively inhibited, and the purpose of treating liver fibrosis is achieved. The GSTM3 inhibitor comprises one or both of a reagent for reducing expression of GSTM3 and a regulator for reducing product of GSTM3. The reagent for reducing expression of GSTM3 comprises a reagent for knocking down or silencing GSTM3; and the regulator for reducing product of GSTM3 comprises a GSTM3 antibody or a protease for degrading the product of GSTM3. The reagent for knocking down or silencing GSTM3 comprises siRNA, shRNA or miRNA. The siRNA comprises a sense strand and an antisense strand, wherein the sequence of the sense strand is shown in SEQ ID NO. 1, and the sequence of the antisense strand is shown in SEQ ID NO. 2. The siRNA can successfully reduce the expression level of GSTM3.

[0028] The application provides a medicine for treating liver fibrosis, wherein the medicine comprises siRNA, and the siRNA comprises a sense strand and an antisense strand, wherein the sequence of the sense strand is shown in SEQ ID NO. 1, and the sequence of the antisense strand is shown in SEQ ID NO. 2.

[0029] In the present application, the drug also includes pharmaceutically acceptable adjuvants, such as excipients, preservatives or antioxidants, etc. The drug can use the above-mentioned siRNA as the only active ingredient for treating liver fibrosis, or can combine other active ingredients for treating liver fibrosis to treat liver fibrosis together. The research results of the present application show that using the above-mentioned siRNA as the only active ingredient can effectively treat liver fibrosis.

[0030] The present application provides a system for predicting or diagnosing liver fibrosis reversal, comprising a data processing device and a detection device, wherein the detection device is configured to detect the expression level of GSTM3 in a sample to be tested and a sample of a liver fibrosis patient; the data processing device comprises a conclusion output module, and if the expression level of GSTM3 in the sample to be tested is significantly reduced compared with the expression level of GSTM3 in the sample of the liver fibrosis patient, the sample to be tested is predicted or diagnosed to have occurred fibrosis reversal; the sample to be tested is a liver fibrosis patient who is receiving drug treatment.

[0031] In the present application, the data processing device further comprises a data input module, a data recording module and a data comparison module; the data input module is configured to input the expression level of GSTM3 in the sample to be tested and the sample of the liver fibrosis patient; the data recording module is configured to store the expression level of GSTM3 in the sample to be tested and the expression level of GSTM3 in the sample of the liver fibrosis patient; the data comparison module is configured to receive the expression level of GSTM3 in the sample to be tested and the expression level of GSTM3 in the sample of the liver fibrosis patient sent by the data input module, and compare the expression level of GSTM3 in the sample of the liver fibrosis patient stored in the data recording module with the expression level of GSTM3 in the sample to be tested; the conclusion output module is configured to receive the comparison result sent by the data comparison module, and determine the comparison result according to a predetermined determination condition. The predetermined determination condition is that if the expression level of GSTM3 in the sample to be tested is significantly reduced compared with the expression level of GSTM3 in the sample of the liver fibrosis patient, the sample to be tested has occurred fibrosis reversal; the sample to be tested is a liver fibrosis patient who is receiving drug treatment.

[0032] In the present application, all raw material components are commercially available products well known to those skilled in the art, unless otherwise specified.

[0033] The technical solutions provided by the present application will be described in detail below in combination with embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0034] In the following examples, the mice of the present application are C57BL / 6J male mice, purchased from Beijing Saiye Biotechnology Co., Ltd. They are bred in the animal house of the Beijing Institute of Science and Technology Research Institute for Analysis and Testing, SPF level, temperature maintained at 22±2℃, humidity maintained at 50-60%, and the feeding process follows the living habits of mice, with a light cycle of 12h light and 12h dark.

[0035] 12.5% carbon tetrachloride preparation method: 12.5mL carbon tetrachloride is uniformly mixed with 87.5mL olive oil to obtain 12.5% carbon tetrachloride.

[0036] Example 1

[0037] Use of a reagent for detecting the expression level of GSTM3 in the preparation of a product for predicting or diagnosing the reversal of liver fibrosis

[0038] 1.1, Construction of mouse liver fibrosis model and mouse liver fibrosis reversal model

[0039] The mice were randomly divided into 4 groups, namely the control group (Ctrl), the CCl4 model group (CCl4), the 4-week reversal group (4W), and the 12-week reversal group (12W).

[0040] The control group is a healthy mouse, i.e., a mouse that has not been treated with 12.5% carbon tetrachloride.

[0041] I. Construction of mouse liver fibrosis model:

[0042] The mouse was injected intraperitoneally with 12.5% carbon tetrachloride at a dose of 0.01mL / g twice a week for 8 weeks to obtain a mouse liver fibrosis model (referred to as the CCl4 model group or model group).

[0043] II. Construction of mouse liver fibrosis reversal model:

[0044] (1) Construction of a model that spontaneously reverses for 4 weeks after mouse liver fibrosis (referred to as the 4-week reversal group)

[0045] The mouse was injected intraperitoneally with 12.5% carbon tetrachloride at a dose of 0.01mL / g twice a week for 8 weeks, and then the injection of 12.5% carbon tetrachloride was stopped, and the mouse was allowed to spontaneously reverse for 4 weeks.

[0046] (2) Construction of a model that spontaneously reverses for 12 weeks after mouse liver fibrosis (referred to as the 12-week reversal group)

[0047] The mouse was injected intraperitoneally with 12.5% carbon tetrachloride at a dose of 0.01mL / g twice a week for 8 weeks, and then the injection of 12.5% carbon tetrachloride was stopped, and the mouse was allowed to spontaneously reverse for 12 weeks.

[0048] The liver tissues of the control group, CCl4 model group, reverse 4 weeks group and reverse 12 weeks group were collected respectively, and HE staining and Masson staining were performed, and the inflammation level and liver fibrosis level of each group were analyzed.

[0049] Figure 1 The results of Fig. 2B show that the CCl4 model group mice have extensive collagen fiber deposition, inflammatory cell infiltration, and part of the area has formed a pathological pseudolobular structure, while the control group liver tissue has no similar performance, indicating that the liver fibrosis mouse model is successfully constructed. In the liver fibrosis reversal model mice, the collagen fiber deposition and inflammatory cell infiltration of the reverse 4 weeks group and the reverse 12 weeks group are significantly reduced, and are positively correlated with the reversal period.

[0050] 1.2, the reagent for detecting the expression level of GSTM3 is used for predicting or diagnosing liver fibrosis reversal

[0051] 1.2.1, respectively, from the model group, the reverse 4 weeks group, the reverse 12 weeks group, the serum and liver of each group of mice are randomly analyzed by proteomics.

[0052] Figure 1 The results of Fig. 2A show that compared with the model group, the expression of GSTM3 protein in the serum and liver of each liver fibrosis reversal mouse in the reverse 4 weeks group and the reverse 12 weeks group is significantly down-regulated, and has a significant decreasing trend with the decrease of liver fibrosis.

[0053] 1.2.2, according to the successful construction of the liver protein of the control group, the CCl4 model group, the reverse 4 weeks group and the reverse 12 weeks group of mice in the above 1.1 part, the expression level of GSTM3 is detected by Western blot, and the information of GSTM3 primary antibody is shown in Table 1. At the same time, the expression of GSTM3 protein in the control group, the CCl4 model group, the reverse 4 weeks group and the reverse 12 weeks group of mice constructed in the above 1.1 part is analyzed by immunohistochemistry.

[0054] Figure 1 The results of Fig. 2B show that the expression of GSTM3 in the control group is low, while the expression in the liver tissue of the liver fibrosis model mice is significantly increased; after the liver fibrosis is reversed, the expression level of GSTM3 is significantly reduced, and has a significant decreasing trend with the decrease of liver fibrosis. Western blot analysis further confirms that the expression of GSTM3 protein in the liver tissue of the liver fibrosis model mice is increased, and the expression level of the reverse model tissue is significantly reduced (see Fig. 2C). Figure 1

[0055] Table 1, information of primary antibody

[0056]

[0057] ​

[0058] Figure 1 The results show that the expression level of GSTM3 in the fibrosis model mice is significantly increased compared with the control group, and the expression level of GSTM3 in each mouse of the reverse 4-week group and the reverse 12-week group is significantly decreased compared with the model group.

[0059] Example 2

[0060] Application of reagent for detecting expression level of GSTM3 in preparation of product for diagnosing liver fibrosis or grading liver fibrosis progression

[0061] Plasma samples of 85 liver fibrosis patients and 23 healthy people were collected from Beijing Ditan Hospital, Capital Medical University. Among the 85 patients, 37 were male (43%) and 48 were female (57%), with an average age of 50 years (23-75 years). The liver fibrosis patients were diagnosed by liver biopsy and scored by the modified Scheuer scoring system, which includes four aspects: no fibrosis score S0, enlarged portal area score S1, enlarged portal area fibrosis with a small amount of fibrous septum formation score S2, a large amount of fibrous septum formation with lobular formation, and structure disorder score S3, and possible or definite cirrhosis score S4. According to the modified Scheuer scoring system, the samples were divided into three groups, namely the healthy control (HC) group, the S1-2 group and the S3-4 group. The known liver fibrosis patients were divided into the S1-2 group and the S3-4 group according to the severity of liver fibrosis, and the plasma of the patients in the S1-2 group and the S3-4 group was used as the test sample.

[0062] The clinical relevant information of the collected samples is shown in Table 2.

[0063] Table 2 Clinical relevant information of samples

[0064]

[0065]

[0066] In Table 2, “ / ” indicates that the detection is not collected; and the data in each row of the item names ALT (U / L), AST (U / L), GGT (U / L), ALP (U / L), Glu (mmol / L) and TG (mmol / L) represent the median (25% median, 75% median).

[0067] The expression level of GSTM3 in the plasma samples of each group is detected by an ELISA kit (GSTM3 elisa kit purchased from biorbyt, item number: orb778927). If the expression level of GSTM3 in the sample to be tested is significantly higher than that in the healthy control group, it indicates that the sample has a risk of liver fibrosis. If the expression level of GSTM3 in the sample to be tested is significantly higher than that in the S1-2 group, it indicates that the sample has a liver fibrosis degree of S3-4.

[0068] Figure 2 The results show that the expression level of GSTM3 in the plasma samples of the S1-2 group and the S3-4 group is significantly higher than that in the healthy control group, and the S3-4 group has a significant increasing trend with the aggravation of liver fibrosis compared with the S1-2 group.

[0069] Example 3

[0070] Use of a GSTM3 inhibitor in the preparation of a product for treating liver fibrosis

[0071] 3.1 Method for inducing activation of hepatic stellate cells by TGF-β1:

[0072] (1) Take the logarithmic growth period LX2 cells (recorded as LX2 cells in the resting state) to prepare cell suspension according to cell subculture, and inoculate 5×10 5 cells per well into a 6-well plate with complete medium added;

[0073] (2) After 12 hours of culture, replace 2% complete medium;

[0074] (3) After 12 hours of starvation, replace 10% complete medium and add 10 ng / mL TGF-β1 for stimulation;

[0075] (4) After 12 hours of culture, collect the cells to obtain activated LX2 cells (recorded as TGF-β).

[0076] 2.2 siRNA-GSTM3 transfection of LX2 cells, steps as follows:

[0077] (1) Take the logarithmic growth period LX2 cells, digest to prepare single cell suspension, and inoculate 5×10 5 cells per well into a 6-well plate with complete medium added, and incubate in a 37℃, 5% CO2 incubator overnight to make the cell density reach about 50% on the next day of transfection;

[0078] (2) For each well of cells, add siRNA for transfection, and after 12 hours of serum starvation treatment, continuously culture for 24 hours to obtain GSTM3 knockout cells (recorded as si-GSTM3);

[0079] The siRNA sequence is: sense strand, 5'-GACAUCAUAGAGAACCAAGUATT-3' (SEQ ID NO. 1); antisense strand, 5'-UACUUGGUUCUCUAUGAUGUCTT-3' (SEQ ID NO. 2).

[0080] The activated GSTM3 knockout cells (denoted as TGF-β+si-GSTM3) were obtained by giving the GSTM3 knockout cells 10 ng / mL TGF-β1 stimulation for 48 hours.

[0081] The RNA and protein of the GSTM3 knockout cells or the quiescent LX2 cells (Ctrl) were extracted for Realtime PCR and Western blot detection of the GSTM3 protein expression, respectively.

[0082] In the Realtime PCR detection, the Realtime PCR primer sequences used are shown in Table 3, and after the Realtime PCR reaction system is prepared according to Table 4, the amplification is performed according to the Realtime PCR reaction program of Table 5.

[0083] Table 3 Realtime PCR primer sequences

[0084]

[0085] Table 4 Realtime PCR reaction system

[0086] Reagent Dose (mL) PowerUp SYBR Green Master Mix (2x) 5 Sterile, nuclease-free water 2.5 Upstream primer 0.25 Downstream primer 0.25

[0087] Table 5 Green Realtime PCR reaction program

[0088] Step Temperature (°C) Time Number of cycles Pre-denaturation 95 5 min 1 Denaturation 95 15 sec 40 Annealing 60 1 min 40 Extension 72 30 sec 40

[0089] Figure 3 The results of A-B in Table 1 show that, compared with the control group (LX2 cells, Ctrl), the mRNA and protein expression levels of GSTM3 in the GSTM3 knockout cells are significantly reduced, indicating that the GSTM3 knockout cell (si-GSTM3) model is successfully constructed.

[0090] The Western blot was performed on the GSTM3 knockout cells or the quiescent LX2 cells (NC) to detect the expression levels of GSTM3, COL1A1, Fibronectin and TIMP1, wherein the primary antibody information of GSTM3, COL1A1, Fibronectin and TIMP1 is shown in Table 1.

[0091] Western blot analysis confirmed that GSTM3 knockout could down-regulate the protein expression levels of fibrosis markers COL1A1, Fibronectin and TIMP1 in quiescent LX-2 cells (see Figure 3 C) in Example 1.

[0092] The RNA was extracted from activated GSTM3 knockout cells, quiescent LX2 cells (Ctrl) or activated LX2 cells, and the mRNA levels of COL1A1, TIMP1 and a-SMA were detected by the Realtime PCR method described above.

[0093] Figure 3 The results of D in Example 1 show that GSTM3 knockout can significantly inhibit the mRNA levels of a-SMA, TIMP1 and COL1A1 induced by TGF-β.

[0094] Western blot was performed to detect the expression levels of Elastin, COL1A1, Fibronectin and TIMP1 in activated GSTM3 knockout cells, quiescent LX2 cells (Ctrl) or activated LX2 cells, wherein the primary antibody information of Elastin, COL1A1, Fibronectin and TIMP1 is shown in Table 1.

[0095] Figure 3 The results of E in Example 1 confirm that GSTM3 knockout can significantly inhibit the expression levels of COL1A1, Fibronectin and Elastin induced by TGF-β.

[0096] In summary, GSTM3 knockout can inhibit the activation of hepatic stellate cells, thereby achieving the purpose of treating liver fibrosis.

[0097] 3.2 GSTM3 overexpression can promote the activation of hepatic stellate cells

[0098] GSTM3 overexpression vector was constructed and transfected into LX-2 cells to obtain GSTM3 overexpression cells, which were serum-starved for 12 hours and then cultured for 24 hours. The cells were harvested and subjected to Western blot analysis.

[0099] Figure 4 The results of A in Example 2 show that the expression level of GSTM3 in GSTM3 overexpression cells is significantly higher than that in the control group (LX-2 cells).

[0100] Figure 4The results of B in the figure showed that compared with the control group (LX-2 cells), the expression of fibrosis markers α-SMA, COL1A1 and TIMP1 in the GSTM3 overexpression group (OE-GSTM3) was significantly up-regulated, and GSTM3 overexpression could promote the activation of hepatic stellate cells.

[0101] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can also be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. The application of a reagent for detecting GSTM3 protein expression levels in the preparation of products for predicting or diagnosing liver fibrosis reversal.

2. The application according to claim 1, characterized in that, The reagents for detecting GSTM3 protein expression levels include GSTM3 antibodies or primer pairs for detecting GSTM3 expression; the sequences of the primer pairs for detecting GSTM3 expression are shown in SEQ ID NO. 3 to SEQ ID NO.

4.

3. The application of a reagent for detecting GSTM3 protein expression levels in the preparation of products for predicting or diagnosing liver fibrosis or grading the progression of liver fibrosis.

4. The application of a GSTM3 inhibitor in the preparation of a product for treating liver fibrosis, characterized in that, The GSTM3 inhibitor is a reagent that reduces GSTM3 expression. The reagent that reduces GSTM3 expression is a reagent that knocks down or silences GSTM3. The reagent that knocks down or silences GSTM3 is siRNA. The siRNA consists of a sense strand and an antisense strand, the sequence of which is shown in SEQ ID NO.1 and the sequence of which is shown in SEQ ID NO.

2.

5. A system for predicting or diagnosing the reversal of liver fibrosis, characterized in that, The device includes a data processing unit and a detection unit. The detection unit is used to detect the expression level of GSTM3 protein in a test sample and a liver fibrosis patient sample. The data processing unit includes a conclusion output module. If the expression level of GSTM3 protein in the test sample is significantly lower than that in the liver fibrosis patient sample, the test sample is predicted or diagnosed as having undergone fibrosis reversal. The test sample is a liver fibrosis patient receiving drug treatment.

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