Application of rhizoma coptidis alkaloid in preparation of medicine for down-regulating liver TREM2 expression

Abstract

The invention relates to application of coptis alkaloid in preparation of a medicine for down-regulating liver TREM2 expression, and belongs to the technical field of biological medicine. Animal experiments prove that the rhizoma coptidis alkaloid has the effects of improving liver pathological injury, lipid droplet deposition, liver function impairment, fatty degeneration, iron overload and inflammatory response caused by MASH, and meanwhile, the expression level of TREM2 is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of coptis alkaloids in the preparation of drugs for downregulating liver TREM2 expression. Background Art

[0002] Metabolic-associated fatty liver disease (MAFLD) is a metabolic stress-induced liver injury that is closely related to insulin resistance and genetic susceptibility. It is characterized by diffuse hepatocyte steatosis, accompanied by chronic inflammation and apoptosis of hepatocytes. The disease spectrum includes metabolic-associated fatty liver, metabolic-associated steatohepatitis (MASH), cirrhosis, and hepatocellular carcinoma. MAFLD not only causes liver damage, but is also closely related to the high incidence of metabolic syndrome, type 2 diabetes (T2DM), atherosclerotic cardiovascular disease, and colorectal tumors. With the prevalence of obesity and metabolic syndrome, MAFLD has become the number one chronic liver disease in my country and the primary cause of abnormal liver biochemical indicators in health examinations.

[0003] Triggering receptor expressed on myeloid cells-2 (TREM2) is considered to be a major pathology-induced immune signaling center and plays an important role in myeloid cell activation and survival. TREM2 expression in macrophages is believed to be associated with chronic inflammation in MAFLD. TREM2 overexpression in liver macrophages of MASH mice promotes the clearance of apoptotic hepatocytes by macrophages, avoids the accumulation of apoptotic hepatocytes in the liver, and induces chronic inflammation. At the same time, it should be noted that TREM2 overexpression in liver macrophages can promote their clearance of red blood cells, induce macrophage iron overload, and aggravate liver inflammatory response. Therefore, liver TREM2 overexpression has a "double-edged sword" effect. In the liver of MASH, the clearance of red blood cells by macrophages is the main manifestation of iron overload and chronic inflammation. At present, most studies on TREM2 in MASH focus on the role of TREM2-mediated hepatic macrophages in phagocytosing apoptotic hepatocytes to eliminate inflammation, while ignoring its role in phagocytizing red blood cells to aggravate inflammation.

[0004] At present, there is no literature reporting whether Coptis chinensis alkaloids are correlated with liver TREM2 expression. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and use coptis alkaloids that can downregulate liver TREM2 expression in the preparation of drugs for downregulating liver TREM2 expression.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides the use of Coptis chinensis alkaloids in the preparation of a drug for downregulating liver TREM2 expression.

[0008] The present invention confirms through animal experiments that Coptis chinensis alkaloids have the effect of improving liver pathological damage, lipid droplet deposition, liver function damage, fatty degeneration, iron overload and inflammatory response caused by MASH, while increasing the expression level of TREM2.

[0009] As a preferred embodiment of the application of the present invention, the active ingredients in the Coptis chinensis alkaloids include at least one of tertrenol, epiberberine, coptis chinensis, palmatine and berberine.

[0010] Liquid chromatography detection revealed that the coptis alkaloids of the present invention contained biologically active ingredients such as tertrenol, epiberberine, coptis pine, palmatine and berberine.

[0011] As a preferred embodiment of the application of the present invention, the mass ratio of the tetrandrine, epiberberine, coptisine, palmatine and berberine is tetrandrine: epiberberine: coptisine: palmatine: berberine = (2.50-4.20): (5.50-7.40): (13.10-15.30): (7.60-9.30): (55.10-72.30).

[0012] As a preferred embodiment of the application of the present invention, the mass ratio of the tetrandrine, epiberberine, coptisine, palmatine and berberine is tetrandrine: epiberberine: coptisine: palmatine: berberine = 3.07:6.75:14.07:8.42:67.68.

[0013] As a preferred embodiment of the application of the present invention, the extraction method of the coptis alkaloids comprises: adding 10 times the amount of 1.5v / v% sulfuric acid solution to the coptis chinensis slices, decocting 3 times, each time for 40 minutes; combining the coptis chinensis extracts, adjusting the pH to 5-6 with solid calcium hydroxide, filtering, adding hydrochloric acid to the filtrate to adjust the pH to 1.5-2, adding 5wt% sodium chloride solution to dissolve, standing in a refrigerator overnight, filtering, vacuum drying the filter residue at 50°C, collecting the precipitate, and the obtained precipitate is the coptis chinensis alkaloids.

[0014] As a preferred embodiment of the application of the present invention, the drug for downregulating liver TREM2 expression is a drug for inhibiting liver iron overload and inflammatory response.

[0015] As a preferred embodiment of the application of the present invention, the drug for downregulating liver TREM2 expression is for downregulating liver TREM2 expression in metabolism-related fatty liver disease.

[0016] As a preferred embodiment of the application of the present invention, the metabolism-related fatty liver disease includes metabolism-related fatty liver and / or metabolism-related fatty liver hepatitis.

[0017] As a preferred embodiment of the application of the present invention, the dosage form of the drug includes at least one of an oral preparation, an injection, an inhalation and an external preparation.

[0018] As a preferred embodiment of the application of the present invention, the content of the coptis alkaloid in the drug for downregulating liver TREM2 expression is 25-100 mg / g.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention proposes for the first time that Coptis chinensis alkaloids have the effect of downregulating liver TREM2 expression, can inhibit liver iron overload and inflammatory response of metabolism-related fatty liver disease, and provide a new prevention and treatment approach for metabolism-related fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a liquid chromatogram of the coptis alkaloids of the present invention;

[0022] Figure 2 The effect of coptis alkaloids on the liver appearance of MASH rats in the experimental example of the present invention;

[0023] Figure 3 The effects of coptis alkaloids on liver, liver index, epididymal and perirenal fat in MASH rats in the experimental examples of the present invention;

[0024] Figure 4 The effect of coptis alkaloids on liver histopathology of MASH rats in the experimental examples of the present invention;

[0025] Figure 5 The effect of coptis alkaloids on liver histopathology of MASH rats in the experimental examples of the present invention;

[0026] Figure 6 This is the effect of coptis alkaloids on lipid droplet deposition in liver tissue of MASH rats in the experimental example of the present invention;

[0027] Figure 7 This is the application of coptis alkaloids on liver function of MASH rats in the experimental examples of the present invention;

[0028] Figure 8 This is the effect of coptis alkaloids on lipid accumulation in MASH rats in the experimental examples of the present invention;

[0029] Fig. 9 The effect of coptis alkaloids on iron deposition and inflammation in liver tissue of MASH rats in the experimental example of the present invention;

[0030] Fig.10 The effect of coptis alkaloids on macrophage polarization markers in liver tissue of MASH rats in the experimental examples of the present invention;

[0031] Fig.11 The effect of coptis alkaloids on macrophage polarization markers in liver tissue of MASH rats in the experimental examples of the present invention;

[0032] In the above figures, those marked with "*" indicate significant differences compared with the MASH group, where "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001; those marked with "#" indicate significant differences compared with the Control group, where "#" indicates P < 0.05, "#" indicates P < 0.01, and "#" indicates P < 0.001. DETAILED DESCRIPTION

[0033] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0034] Unless otherwise specified, the reagents and consumables used in the following experimental examples can be obtained through commercial channels.

[0035] The coptis alkaloids used in the following experimental examples can be obtained through commercial channels or extracted according to existing extraction methods. The coptis alkaloids used in this experimental example are mainly extracted according to the following literature:

[0036] Tu Yaosheng, Jiang Zhiqiang, Sun Dongmei, Zhang Jianjun, Li Shaolin, Fu Jianwu. Study on the extraction and purification process of Coptis chinensis alkaloids, Chinese Medicine New Drugs and Clinical Pharmacology, 2012, 23(2): 208-212.

[0037] The extraction method in the literature was improved, specifically as follows: adding 10 times the amount of 1.5v / v% sulfuric acid solution to the Coptis chinensis slices, decocting 3 times, each time for 40 minutes; combining the Coptis chinensis extracts, adjusting the pH to 5-6 with solid calcium hydroxide, filtering, adding hydrochloric acid to the filtrate to adjust the pH to 1.5-2, adding 5wt% sodium chloride solution to dissolve, standing in a refrigerator overnight, filtering, vacuum drying the filter residue at 50°C, collecting the precipitate, and the obtained precipitate is Coptis chinensis alkaloids.

[0038] SPF male Sprague Dawley rats used in the following experimental examples were purchased from Guangdong Medical Laboratory Animal Center. Rats were kept in a standard animal room with a temperature of 23±2°C and a humidity of 50±10%, a 12h / 12h (lighting on at 8:00) circadian rhythm, and free access to food. This experiment was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals.

[0039] The experimental steps in the following experimental examples without special remarks are all commonly used experimental operations in this field, and reference may be made to "Pathology Experiment Guide" (2014, Science Press) and the like.

[0040] In the following experimental examples, the levels of serum ALT and AST and the contents of liver TC, TG, TNF-α and IL-6 were determined using biochemical detection kits purchased from Nanjing Jiancheng Bioengineering Institute. The specific operation steps were carried out according to the kit instructions.

[0041] In the following experimental examples, the protein expression of liver TREM2, F4 / 80 and CD11c was detected by immunofluorescence. F4 / 80 and CD11c antibodies were purchased from Wuhan Saiweier Biotechnology Co., Ltd., and Trem2 antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd.

[0042] The specific operation steps of immunofluorescence are as follows: collect liver tissue for experiment, fix it with 4% paraformaldehyde, and make paraffin sections; slice and dry the slices, and then dewax them to water as usual. After TBS rinsing, repair the antigen and block it with 5wt% BSA; add the primary antibody (TREM2, F4 / 80 and CD11c) diluted in a certain proportion to the slice; after incubation overnight, rinse with TBS, then add fluorescent secondary antibody for incubation, and rinse with TBS after completion. After adding DAPI working solution to incubate and stain the nucleus, wash the cells with TBS. Add fluorescence quencher to seal the slice and observe under a fluorescence microscope.

[0043] Scoring criteria for hepatocyte fatty degeneration: 0 points for hepatocyte fatty degeneration <5%, 1 point for hepatocyte fatty degeneration 5-33%, 2 points for hepatocyte fatty degeneration 34-66%, and 3 points for hepatocyte fatty degeneration >66%.

[0044] Scoring criteria for liver lobular inflammation (counting necrotic foci under a 20x magnification objective): 0 points for no necrotic foci, 1 point for <2 necrotic foci, 2 points for 2-4 necrotic foci, and 3 points for >4 necrotic foci.

[0045] Scoring criteria for hepatocellular ballooning: no hepatocellular ballooning was 0 points, rare hepatocellular ballooning was 1 point, and frequent hepatocellular ballooning was 2 points.

[0046] Evaluation criteria for MASLD activity score (MAS): The sum of the scores of liver fatty degeneration, lobular inflammation and hepatocellular ballooning is MAS. MAS < 3 points can exclude MASH, and MAS > 5 points can be diagnosed as MASH.

[0047] Experimental Example 1

[0048] In order to verify the main active ingredients of Coptis chinensis alkaloids, liquid chromatography was used for qualitative and quantitative analysis. The liquid chromatography conditions are shown in Tables 1 and 2. The results are shown in Figure 1 and Table 3.

[0049] Table 1 Chromatographic conditions

[0050]

[0051] Table 2 Gradient elution conditions

[0052]

[0053] Table 3 Main active ingredients and their relative percentages in Coptis chinensis alkaloids

[0054]

[0055] Experimental Example 2

[0056] In order to verify the effect of coptis alkaloids on the expression of TREM2 in the liver, a metabolism-related fatty hepatitis model was constructed and coptis alkaloids were administered to observe the physiological indicators of the model. The specific scheme is as follows:

[0057] 1 Experimental content

[0058] After one week of adaptive feeding, SPF male Sprague Dawley rats (170±10g) were randomly divided into 7 groups (8 rats in each group), namely: normal group (Normal), model group (MASH), pioglitazone positive drug group (PGZ, 20mg / kg), berberine group (BBR, 60mg / kg), low-dose coptis alkaloid group (25mg / kg CA), medium-dose coptis alkaloid group (50mg / kgCA) and high-dose coptis alkaloid group (100mg / kg CA). Except for the control group, which was given standard feed, the rats in the other groups were fed with high-fat diet (HFD) for 8 consecutive weeks to induce metabolic-related fatty hepatitis model. During the experiment, while modeling, each group of rats was gavaged with blank solution or corresponding drug solution at a volume of 1mL / 100g, and the body weight data of each group of rats were recorded every day. On the day before the experimental sampling, the rats were fasted for 12h, and blood was collected through the abdominal aorta after anesthesia. After blood collection, the rats were euthanized and liver tissues were collected. Serum and liver homogenate supernatant were used for subsequent index detection.

[0059] The observation indicators included liver appearance, coefficients of liver, perirenal fat and epididymal fat, hematoxylin-eosin staining and Oil Red O staining of liver tissue pathological sections, serum ALT and AST levels, liver TC, TG, TNF-α and IL-6 levels, and immunofluorescence detection of TREM2, F4 / 80 and CD11c. The results are shown in Figure 2-8 .

[0060] 2 Experimental results

[0061] (1) Figure 2As shown in the figure, compared with the Control group, the liver tissue in the MASH group was diffusely enlarged, yellow in color, shiny on the surface, and greasy; compared with the MASH group, the pioglitazone group, berberine group, and coptis alkaloid group all alleviated the macroscopic lesions of the liver tissue to varying degrees.

[0062] The high-dose group of Coptis chinensis alkaloids had the best effect. Figure 3 As shown in the figure, in terms of liver index, epididymal fat and perirenal fat weight ratio, the liver index, epididymal fat and perirenal fat weight ratio of the MASH group rats were significantly increased compared with the normal control group (P<0.01). Compared with the MASH group, the liver index, epididymal fat and perirenal fat weight ratio of the pioglitazone group, berberine group and coptis alkaloid group were all downregulated to varying degrees. The liver index, epididymal fat and perirenal fat weight of the coptis alkaloid group were significantly decreased compared with the model group ( Figure 3 AC), among which 100 mg / kg of coptis alkaloids performed most prominently in regulating liver weight index and fat weight index (P<0.05), indicating that coptis alkaloids inhibited high-fat diet-induced liver hypertrophy and the increase of liver index, epididymal fat and perirenal fat in MASH rats.

[0063] (2) Figure 4 As shown in the figure, H&E stained sections of liver tissue were observed under a microscope. The liver lobules and sinusoids in the liver tissue of rats in the Control group were intact, the hepatic cords around the central vein were arranged radially, the cell size was relatively uniform, and the nucleus was clearly visible. Compared with the Control group, diffuse macrovesicular hepatocyte fatty degeneration was observed in the liver tissue of rats in the MASH group. The cell volume was enlarged, there were fat vacuoles of varying sizes in the cytoplasm, the nucleus was centered or offset, the sinusoids were compressed and narrowed, and there was inflammatory cell infiltration. Figure 5 As shown in the data, compared with those in the Control group, the scores of fatty degeneration, lobular inflammation, and hepatocellular ballooning in the liver tissue of rats in the MASH group and the MAS score were significantly increased (P<0.01); and MAS>5 (the scores of fatty degeneration, lobular inflammation, and hepatocellular ballooning were all>1), which met the evaluation criteria of MASH.

[0064] The liver tissues of rats in the pioglitazone group, berberine group and coptis alkaloid group showed improvement in different degrees of hepatocyte fatty degeneration and inflammatory cell infiltration. The scores of fatty degeneration, lobular inflammation and hepatocyte ballooning in the liver tissues of rats in the 100 mg / kg coptis alkaloid group and the MAS score were significantly lower than those in the MASH group (P<0.01). Compared with the berberine group, the effect of 100 mg / kg coptis alkaloid on improving the pathological changes of liver tissue was better than that in the berberine group (P<0.01). This suggests that coptis alkaloid administration can significantly improve the liver pathological damage of MASH rats.

[0065] (3) Figure 6 As shown in the figure, the Oil Red O-stained sections of liver tissue were observed under a microscope. Compared with the Control group, there were a large number of red-stained lipid droplets in the hepatocytes of rats in the MASH group, and the Oil Red O-stained area of ​​lipid droplets was also significantly increased (P<0.01). However, compared with the rats in the MASH group, the hepatocytes of rats in the pioglitazone group, the berberine group, and the coptis alkaloid group showed different degrees of improvement in red-stained lipid droplets. The red-stained lipid droplet area in the 100 mg / kg coptis alkaloid group was significantly reduced compared with the model group (P<0.01) and was better than the berberine group, indicating that coptis alkaloid administration improved lipid droplet deposition in the liver tissue of MASH rats.

[0066] (4) Figure 7 As shown in the figure, AST and ALT are one of the important biochemical indicators for evaluating liver function. Compared with the Control group, the serum ALT and AST levels of rats in the MASH group were significantly increased (P<0.01), indicating that the liver function of rats in the MASH group was significantly abnormal. Compared with the MASH group, the serum ALT and AST levels of rats in the pioglitazone group, the coptis group and the coptis alkaloid group were significantly decreased compared with the model group (P<0.01), which improved the abnormal liver function of MASH rats to varying degrees. Among them, 100 mg / kg coptis alkaloid had the best effect in improving ALT and AST, indicating that the administration of coptis alkaloid significantly inhibited the liver function damage of MASH rats.

[0067] (5) Figure 8 As shown in the results, compared with the Control group, the TG and TC contents in the liver tissue of rats in the MASH group were significantly increased (P<0.01), indicating that long-term feeding of a high-fat diet can induce lipid metabolism disorders in the liver of rats. Compared with the MASH group, the lipid metabolism disorders in the liver of rats in the pioglitazone group, the berberine group and the coptis alkaloid group were effectively alleviated. Among them, the three doses of coptis alkaloids can reduce the lipid content, among which 100 mg / kg coptis alkaloids significantly improved the TG and TC contents in the liver tissue of rats (P<0.01), and the effect of reducing the lipid content in MASH rats was the best. It shows that coptis alkaloids can reduce the lipid content in the body and alleviate the fatty degeneration of MASH rats.

[0068] (6) Fig. 9As shown in the data, compared with those in the Control group, the iron content and the levels of TNF-α and IL-1β in the liver tissue of the rats in the MASH group were significantly increased, indicating that the liver iron overload and inflammatory response of the MASH rats were aggravated. The iron content and the levels of TNF-α and IL-1β in the liver tissue of the rats in the pioglitazone group, berberine group and coptis alkaloid group were significantly decreased to varying degrees compared with those in the MASH group (P<0.01), among which 100 mg / kg coptis alkaloid improved the iron content and the levels of TNF-α and IL-1β most significantly. The above results indicate that coptis alkaloid can alleviate the liver iron overload and inflammatory response of MASH rats.

[0069] (7) Figure 10-11 As shown, F4 / 80 is a marker of macrophages, CD11c is a marker of M1 macrophage polarization, and immunofluorescence was used to detect the expression of F4 / 80, CD11c and TREM2 in the liver of rats ( Fig.10 ), the expression of F4 / 80, CD11c and TREM2 was almost undetectable in the Control group, while in the MASH group, the positive areas of F4 / 80, CD11c and TREM2 increased significantly, indicating a large number of M1 macrophage infiltration and high expression of TREM2. The infiltration of M1 macrophages in the liver tissue of rats in the pioglitazone group, berberine group and coptis alkaloid group decreased to varying degrees, among which 100 mg / kg coptis alkaloid inhibited F4 / 80, CD11c and TREM2 most significantly, indicating that coptis alkaloid can inhibit the expression of TREM2 in M1 macrophages in the liver of MASH rats ( Fig.11 ).

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Application of Coptis chinensis alkaloids in the preparation of drugs for downregulating liver TREM2 expression.

2. The use according to claim 1, characterized in that The active ingredients in the coptis alkaloids include at least one of tertrenol, epiberberine, coptisine, palmatine and berberine.

3. The use according to claim 2, characterized in that The mass ratio of the tetrandrine, epiberberine, coptisine, palmatine and berberine is tetrandrine: epiberberine: coptisine: palmatine: berberine = (2.50-4.20): (5.50-7.40): (13.10-15.30): (7.60-9.30): (55.10-72.30).

4. The use according to claim 3, characterized in that The mass ratio of the tetrandrine, epiberberine, coptisine, palmatine and berberine is tetrandrine: epiberberine: coptisine: palmatine: berberine=3.07:6.75:14.07:8.42:67.

68.

5. The use according to claim 1, characterized in that The drug for downregulating liver TREM2 expression is a drug for inhibiting liver iron overload and inflammatory response.

6. The use according to claim 1, characterized in that The drug for downregulating liver TREM2 expression is used to downregulate liver TREM2 expression in metabolism-related fatty liver disease.

7. The use according to claim 6, characterized in that The metabolism-related fatty liver disease includes metabolism-related fatty liver and / or metabolism-related steatohepatitis.

8. The use according to claim 1, characterized in that The dosage form of the drug includes at least one of an oral preparation, an injection, an inhalation and an external preparation.

9. The use according to claim 1, characterized in that The content of the coptis alkaloid in the drug for downregulating liver TREM2 expression is 25-100 mg.

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