Application of OPPC combined with γ-Linolenic acid in the preparation of drugs for treating autoimmune liver diseases

Abstract

The present invention belongs to the field of pharmaceutical technology and discloses the use of 1-Oleoyl-2-palmitoy-sn-glycero-3-phosphocholine (OPPC) combined with γ-Linolenic acid in the preparation of a medicament for treating autoimmune liver disease (ALD). The medicament for treating autoimmune liver disease according to the present invention comprises OPPC and / or γ-Linolenic acid, as well as derivatives of OPPC and / or γ-Linolenic acid. By administering the medicament of the present invention to treat MRL / MpJ-Fas<supgt;lpr< / supgt; (MRL / lpr) mice, the plasma metabolic level and liver function of MRL / lpr mice can be improved, providing a new direction for the clinical treatment of autoimmune liver disease.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to the application of 1-Oleoyl-2-palmitoy-sn-glycero-3-phosphocholine (OPPC) combined with γ-Linolenic acid in the preparation of drugs for treating autoimmune liver disease (ALD). Background Art

[0002] The "powerful function" of basophils (Baso) in allergic reactions has been well-known. As a "bridge" connecting innate immunity and adaptive immunity, their role in autoimmune diseases cannot be underestimated. However, there is currently a lack of an ideal strategy to inhibit Baso activation. Autophagy is a highly conserved catabolic process, which is divided into three types according to different action modes: macroautophagy, microautophagy, and chaperone-mediated autophagy. When cells are stimulated by damaging factors, a large amount of misfolded proteins, damaged organelles, etc. accumulate and are degraded by lysosome-mediated degradation to facilitate the maintenance of the cell's own metabolism, which plays an important role in both physiological and pathological states of the body. There are multi-level cross-regulations between the autophagy signaling pathway and the Baso activation signaling pathway.

[0003] Autoimmune liver disease (ALD) is a liver disease caused by abnormal immune responses of the human body. The pathogenesis of autoimmune liver disease is not fully understood and may be related to genetic, drug, infectious, and environmental factors, etc. However, abnormal immune responses in the human body can cause liver cell damage, produce autoantibodies, and may lead to serious complications such as liver cirrhosis and liver cancer. The treatment of autoimmune liver disease mainly uses drugs such as immunosuppressants and steroid hormones to regulate the immune system, reduce liver damage, and control the inflammatory response and disease progression. In addition, it is necessary to adjust the diet and lifestyle according to individual circumstances, and strengthen medical follow-up and daily health care to avoid exacerbating the condition or developing complications. Therefore, finding new targets with significant efficacy and high safety has become an urgent problem to be solved in the current treatment of autoimmune liver disease. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of OPPC combined with γ-Linolenic acid in the preparation of drugs for treating autoimmune liver disease.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a drug for treating autoimmune liver disease, comprising OPPC and / or γ-Linolenic acid, or derivatives of OPPC and / or γ-Linolenic acid.

[0007] The present invention constructs MRL / MpJ-Fas lpr (MRL / lpr) mice with basophil autophagy deficiency, and finds that basophil autophagy deficiency can improve the metabolic level and liver function of MRL / lpr mice. Plasma of MRL / lpr mice was collected for non-targeted metabolomics detection, and it was found that the expression of OPPC and γ-Linolenic acid was significantly increased in the basophil autophagy deficiency group, and was positively correlated with plasma metabolic level and liver function. The results of drug administration treatment of OPPC and γ-Linolenic acid in MRL / lpr mice showed that: the drug administration treatment of OPPC and γ-Linolenic acid could improve the plasma metabolic level and liver function of MRL / lpr mice, and the drug administration treatment effect of OPPC combined with γ-Linolenic acid was significantly better than that of single drug administration treatment.

[0008] As a preferred embodiment of the drug according to the present invention, it further comprises a pharmaceutically acceptable carrier or excipient.

[0009] As a preferred embodiment of the drug according to the present invention, the drug is an oral preparation or an injection preparation.

[0010] In a second aspect, the present invention applies OPPC and / or γ-Linolenic acid in the preparation of a drug for preventing and / or treating autoimmune liver disease.

[0011] As a preferred embodiment of the application according to the present invention, when the drug is administered, the dose of OPPC and / or γ-Linolenic acid is 10 mg / kg to 20 mg / kg once, every 1 to 2 days.

[0012] As a preferred embodiment of the application according to the present invention, the drug is an oral preparation or an injection preparation.

[0013] As a preferred embodiment of the application according to the present invention, the functions of the drug are at least one of the following:

[0014] Ⅰ. Reduce the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, cholesterol, and low-density lipoprotein in plasma;

[0015] Ⅱ. Increase the levels of high-density lipoprotein and A / G ratio in plasma;

[0016] III. Improve the level of hepatocyte degeneration and necrosis.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention discovers that autophagy deficiency of basophils can improve the metabolic level and liver function of MRL / lpr mice. Collecting the plasma of MRL / lpr mice for non-targeted metabolomics detection reveals that the expressions of OPPC and γ-Linolenic acid are significantly increased in the autophagy deficiency group of basophils, and are positively correlated with the plasma metabolic level and liver function. The drugs of the present invention include OPPC and / or γ-Linolenic acid. The results of administering drugs to treat MRL / lpr mice show that the administration of OPPC and γ-Linolenic acid can improve the plasma metabolic level and liver function of MRL / lpr mice, and the therapeutic effect of the combined administration of OPPC and γ-Linolenic acid is significantly better than that of single administration, providing a new direction for the clinical treatment of autoimmune liver diseases. Description of the Drawings

[0019] Figure 1 It is the plasma biochemical test results of MRL / lpr mice with autophagy deficiency of basophils; in the figure, A is the test result of the plasma alanine aminotransferase (ALT) level of MRL / lpr mice, B is the test result of the plasma aspartate aminotransferase (AST) of MRL / lpr mice, C is the test result of the plasma lactate dehydrogenase (LDH) of MRL / lpr mice, D is the test result of the plasma creatine kinase (CK) of MRL / lpr mice, E is the test result of the plasma α-hydroxybutyrate dehydrogenase (α-HBDH) of MRL / lpr mice, F is the test result of the plasma cholesterol of MRL / lpr mice, G is the test result of the plasma high-density lipoprotein of MRL / lpr mice, H is the test result of the plasma low-density lipoprotein of MRL / lpr mice, I is the test result of the plasma bile acids of MRL / lpr mice, J is the plasma albumin / globulin (A / G) ratio of MRL / lpr mice; P<0.05 indicates significant difference, marked with *.

[0020] Figure 2Non-targeted metabolomics detection results of plasma from MRL / lpr mice with basophil autophagy deficiency; in the figure, A shows the PCA analysis of the differences between the two groups of samples, with green representing the Control group and red representing the basophil autophagy deficiency group. B shows the volcano plot analysis of the differential metabolites between the two groups of samples. The selection criteria for differential metabolites are that the variable importance in the projection (VIP) is greater than 1 and the P-value is less than 0.05. C-G show the violin plot analysis of the differential metabolites between the two groups of samples. The box in the center of the violin plot represents the interquartile range, the thin black lines extending from it represent the 95% confidence interval, the black horizontal line in the middle corresponds to the median, and the outer shape depicts the data distribution density.

[0021] Figure 3 Correlation analysis between differential metabolites in plasma and plasma biochemical indexes of MRL / lpr mice with basophil autophagy deficiency; in the figure, A shows the RDA analysis of differential metabolites and enzymes, and B shows the RDA analysis of differential metabolites and proteins and lipids. The length of the arrow represents the magnitude of the correlation between the disease severity index and the sample distribution. The longer the line, the greater the correlation. The angle between the arrow and the sorting axis represents the correlation, with an acute angle indicating a positive correlation and an obtuse angle indicating a negative correlation.

[0022] Figure 4 Biochemical detection results of plasma from MRL / lpr mice treated with OPPC and γ-Linolenic acid; in the figure, A shows the detection results of the alanine aminotransferase (ALT) level in the plasma of MRL / lpr mice, B shows the detection results of the aspartate aminotransferase (AST) in the plasma of MRL / lpr mice, C shows the detection results of the lactate dehydrogenase (LDH) in the plasma of MRL / lpr mice, D shows the detection results of the creatine kinase (CK) in the plasma of MRL / lpr mice, E shows the detection results of cholesterol in the plasma of MRL / lpr mice, F shows the detection results of high-density lipoprotein in the plasma of MRL / lpr mice, G shows the detection results of low-density lipoprotein in the plasma of MRL / lpr mice, and H shows the albumin / globulin (A / G) ratio in the plasma of MRL / lpr mice; P<0.05 indicates significant difference, marked with *.

[0023] Figure 5 H&E staining detection results of liver pathology of MRL / lpr mice treated with OPPC and γ-Linolenic acid. Detailed implementation methods

[0024] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0026] Example 1: Biochemical detection of plasma in basophil autophagy-deficient MRL / lpr mice

[0027] Autophagy gene Atg5 knockout mice (Atg5 flox / flox Cre + / - , Atg5 - / - ) and autophagy gene Atg5 non-knockout control mice (Atg5 flox / flox Cre - / - , Atg5 + / + ) were provided by Shanghai Model Organisms Center, Inc. The basophil autophagy-deficient mouse model was constructed, specifically referring to the Chinese invention patent document CN 118120702B (Construction method and application of a basophil autophagy-deficient mouse).

[0028] The mice were grouped as follows:

[0029] The Control group was MRL / lpr mice without any intervention; the Atg5 - / - Baso adoptive transfer group was basophil autophagy-deficient mice; the Atg5 + / + Baso adoptive transfer group was autophagy gene Atg5 non-knockout control mice that received adoptive transfer of basophils; at the same time, MRL / MpJ mice were set as the normal control group for MRL / lpr lupus mice.

[0030] The plasma of mice in each group was collected respectively, and the biochemical metabolic level of mouse plasma was detected using an automatic biochemical analyzer (Roche, Switzerland), and the data were statistically analyzed using Graphpad Prism 8.0.2 software.

[0031] The experimental results are shown in Figure 1 , in the plasma enzyme test results, the Atg5 - / - Baso adoptive transfer group was compared with the model Control group and Atg5 + / +In the Baso adoptive transfer group, there was an obvious decreasing trend in plasma ALT, AST, and LDH, reflecting the improvement of its liver metabolism level; plasma CK showed a decreasing trend, reflecting the alleviation of muscle tissue damage; no obvious change was found in α-HBDH. The above results indicate that Baso autophagy deficiency improves the levels of enzymes in plasma biochemical metabolism of lupus mice.

[0032] In the test results of plasma proteins and lipids, Atg5 - / - In the Baso adoptive transfer group compared with the model Control group and Atg5 + / + In the Baso adoptive transfer group, the levels of plasma cholesterol, bile acid, and low-density lipoprotein decreased, and there was no obvious change in high-density lipoprotein; plasma A / G was improved. In biochemical metabolism, high-density lipoprotein can transport cholesterol in the body to various organs, improve immunity, and prevent atherosclerosis. While low-density lipoprotein will cause further deposition of cholesterol in the body within the blood vessel wall, exacerbating the occurrence of atherosclerosis. Bile acid is the end product of the metabolism of cholesterol in the liver of the body. In the biochemical metabolism test, the levels of cholesterol, bile acid, and low-density lipoprotein in the plasma of Baso autophagy-deficient lupus mice decreased, and the level of high-density lipoprotein did not change significantly; plasma albumin increased, globulin decreased, and the A / G ratio was improved, indicating that Baso autophagy deficiency improved the in vivo metabolism level. The above results indicate that Baso autophagy deficiency improves the in vivo protein and lipid metabolism levels.

[0033] Example 2: Correlation analysis of plasma differential metabolites and plasma biochemical indexes in basophil autophagy-deficient MRL / lpr mice

[0034] (1) Plasma untargeted metabolomics detection of basophil autophagy-deficient MRL / lpr mice

[0035] Take the plasma of basophil autophagy-deficient MRL / lpr mice in Example 1 and send it to Wuhan Metware Biotechnology Co., Ltd. for plasma untargeted metabolomics detection.

[0036] (2) Correlation analysis of plasma differential metabolites and plasma biochemical indexes in basophil autophagy-deficient MRL / lpr mice

[0037] Plasma untargeted metabolomics detection results Figure 2 , OPLS-DA analysis revealed significant differences between the control group (basophil autophagy activation group under autoimmune disease state) and the Atg5KO group (basophil autophagy deficiency group under autoimmune disease state) ( Figure 2 A), volcano plot analysis showed that 66 metabolites were upregulated and 33 metabolites were downregulated in the Atg5KO group ( Figure 2 B).

[0038] Violin metabolite difference analysis showed that the levels of γ-Linolenic acid and OPPC increased in the Atg5KO group, while the levels of 2-Methylsuccinic acid, 2-Hydroxyisophthalic acid, and Arginine decreased relative to the control group ( Figure 2 C–G).

[0039] Example 3: Detection of disease progression in MRL / lpr mice administered with OPPC and γ-Linolenic acid

[0040] To evaluate the effects of OPPC and γ-Linolenic acid on the disease progression of MRL / lpr mice, MRL / lpr mice were divided into: Control group (lupus control without any intervention), γ-Linolenic acid single administration group, OPPC single administration group, OPPC combined with γ-Linolenic acid administration group, and MRL / MpJ mice were set as the normal control group for MRL / lpr mice.

[0041] Among them, in the γ-Linolenic acid single administration group, mice were orally administered γ-Linolenic acid with a vehicle every other day starting from the 8th week, at a dose of 20 mg / kg. In the OPPC single administration group, mice were orally administered γ-Linolenic acid with a vehicle every other day starting from the 8th week, at a dose of 20 mg / kg. In the OPPC combined with γ-Linolenic acid administration group, γ-Linolenic acid and OPPC were evenly mixed, and mice were orally administered with a vehicle every other day starting from the 8th week, with a total dose of 20 mg / kg.

[0042] (1) Plasma biochemical indexes (ALT, AST, LDH, CK, Cholesterol, High-density lipoprotein, Low-density lipoprotein, A / G) of MRL / lpr mice administered with OPPC and γ-Linolenic acid were detected by an automatic biochemical analyzer. The detection method of plasma samples of MRL / lpr mice was the same as that in Example 1.

[0043] (2) H&E detection of liver pathology of MRL / lpr mice administered with OPPC and γ-Linolenic acid

[0044] Deparaffinization: Place the liver tissue slides in xylene I for 20 min and in xylene II for 20 min; place the slides in absolute ethanol I for 5 s, in absolute ethanol II for 5 s → in 95% ethanol I for 5 s, in 95% ethanol II for 5 s → wash with water for 15 s; place the slides in hematoxylin staining solution and stain for 2 min → wash with water for 15 s × 2 → stain with 1% hydrochloric acid alcohol for 3 s → wash with water for 15 s → stain with 1% ammonia water for 3 s → wash with water for 15 s; place the slides in eosin staining solution and stain for 2 min → wash with water for 15 s → place in absolute ethanol for 2 s → dry by baking, and seal with neutral resin; take pictures.

[0045] The experimental results are shown in Figures 4 - 5 : In Figure 4Among them, the plasma ALT, AST, and LDH levels in the single-dose γ-Linolenic acid group and the single-dose OPPC group were significantly lower than those in the Control group, but there was no significant change in CK. The Cholesterol and Low-density lipoprotein levels decreased, while the High-density lipoprotein and A / G increased. In the OPPC combined with γ-Linolenic acid administration group, compared with the single-dose γ-Linolenic acid group and the single-dose OPPC group, the plasma ALT, AST, and LDH levels were further significantly decreased, the Cholesterol and Low-density lipoprotein levels were further significantly decreased, and the High-density lipoprotein and A / G were further significantly increased. In biochemical metabolism, High-density lipoprotein can transport Cholesterol in the body to various organs, improve immunity, and prevent atherosclerosis. While Low-density lipoprotein can cause further deposition of Cholesterol in the body within the blood vessel wall, exacerbating the occurrence of atherosclerosis. In the biochemical metabolism test, in MRL / lpr mice administered with OPPC combined with γ-Linolenic acid, the plasma Cholesterol and Low-density lipoprotein levels decreased, the High-density lipoprotein level increased, and the A / G ratio improved, indicating that the administration of OPPC combined with γ-Linolenic acid improved the body's metabolic level. In Figure 5 Among them, the levels of hepatocyte degeneration and necrosis in the single-dose γ-Linolenic acid group and the single-dose OPPC group were improved compared with the Control group, and in the OPPC combined with γ-Linolenic acid administration group, compared with the single-dose γ-Linolenic acid group and the single-dose OPPC group, the levels of hepatocyte degeneration and necrosis were further significantly improved.

[0046] In summary, compared with the Control group, the plasma biochemical metabolism levels and liver pathology in the γ-Linolenic acid single administration group and the OPPC single administration group were improved, indicating that γ-Linolenic acid or OPPC has an improvement effect on disease progression. However, it is worth noting that the disease progression in the OPPC combined with γ-Linolenic acid administration group was more significantly improved compared with the γ-Linolenic acid single administration group and the OPPC single administration group, indicating that the therapeutic effect of OPPC combined with γ-Linolenic acid administration is significantly better than single treatment. The above results indicate that OPPC combined with γ-Linolenic acid administration can significantly improve the disease progression in MRL / lpr mice, providing a new idea for the treatment of autoimmune liver disease.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

Use of 1.1-Oleoyl-2-palmitoy-sn-glycero-3-phosphocholine (OPPC) as the sole active ingredient in the preparation of a medicament for preventing and / or treating lupus hepatopathy. Use of 1.1-Oleoyl-2-palmitoy-sn-glycero-3-phosphocholine (OPPC) and γ-Linolenic acid in the preparation of a medicament for preventing and / or treating lupus hepatopathy.

3. The application according to claim 1 or 2, characterized in that, The medicament is an oral preparation or an injection preparation.

4. The application according to claim 1 or 2, characterized in that, The functions of the medicament are at least one of the following: Ⅰ. Reducing the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, cholesterol, and low-density lipoprotein in plasma; Ⅱ. Increasing the levels of high-density lipoprotein and A / G ratio in plasma; Ⅲ. Improving the levels of hepatocyte degeneration and necrosis.

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