Application of combination of OPPC and gamma-Linolenic acid in preparation of drugs for treating autoimmune liver diseases

Abstract

The invention belongs to the technical field of medicines, and discloses an application of a combination of 1-Oleoyl-2-palmitoy-sn-glycino-3-phodopholine (OPPC) and gamma-Linolenic acid in preparation of a medicine for treating autoimmune liver diseases (ALD), and a preparation method of the combination of the 1-Oleoyl-2-palmitoy-sn-glycino-3-phodopholine (OPPC) and the gamma-Linolenic acid in the preparation of the medicine for treating the ALD and the application of the combination of the 1-Oleoyl-2-palmitoy-sn-glycino-3-phodopholine (OPPC). The medicine for treating the autoimmune liver diseases, disclosed by the invention, is prepared from OPPC and / or gamma-Linolenic acid, and a derivative of the OPPC and / or the gamma-Linolenic acid. The medicine disclosed by the invention is used for treating MRL / MpJ-Fastlpr (MRL / lpr) mice through administration, so that the plasma metabolism level and the liver function of the MRL / lpr mice can be improved, and a new direction is provided for clinical treatment of the autoimmune liver diseases.

Description

Technical Field

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

[0002] The "powerful function" of basophils (Baso) in allergic reactions is well known. As a "bridge" connecting innate immunity and adaptive immunity, its role in autoimmune diseases cannot be underestimated. However, there is currently a lack of ideal strategies to inhibit Baso activation. Autophagy is a highly conserved catabolic process, which is divided into three types according to different modes of action: macroautophagy, microautophagy, and chaperone-mediated autophagy. When cells are stimulated by damaging factors, a large number of misfolded proteins and damaged organelles accumulate, which are degraded by lysosomes to maintain the cell's own metabolism. This process plays an important role in both physiological and pathological conditions of the body. There are multiple levels of cross-regulation between the autophagy signaling pathway and the Baso activation signaling pathway.

[0003] Autoimmune liver disease (ALD) is a liver disease caused by an abnormal response of the human immune system. The pathogenesis of autoimmune liver disease is not fully understood and may be related to genetics, drugs, infection, and environmental factors. However, abnormal responses of the human immune system can cause damage to liver cells, produce autoantibodies, and may lead to serious complications such as 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 inflammatory responses and disease progression. In addition, it is necessary to adjust diet and lifestyle according to individual conditions, as well as strengthen medical follow-up and daily health care to avoid worsening of the disease or complications. Therefore, finding new targets with significant efficacy and high safety has become an urgent problem to be solved in the 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 a drug 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 a basophil (Baso) autophagy-deficient MRL / MpJ-Fas lpr (MRL / lpr) mice, and found that basophil autophagy deficiency can improve the metabolic level and liver function of MRL / lpr mice. The 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 levels and liver function. The results of the treatment of MRL / lpr mice with OPPC and γ-Linolenic acid showed that the treatment of OPPC and γ-Linolenic acid can improve the plasma metabolic level and liver function of MRL / lpr mice, and the treatment effect of OPPC combined with γ-Linolenic acid is significantly better than that of single treatment.

[0008] As a preferred embodiment of the drug of the present invention, it also includes a pharmaceutically acceptable carrier or excipient.

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

[0010] In a second aspect, the present invention uses 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 of the present invention, when the drug is administered, the dosage of OPPC and / or γ-Linolenic acid is 10 mg / kg to 20 mg / kg once, once every 1 to 2 days.

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

[0013] As a preferred embodiment of the application of the present invention, the function of the drug is 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 level 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 present invention has the following beneficial effects:

[0018] The present invention found that basophil autophagy deficiency can improve the metabolic level and liver function of MRL / lpr mice. The 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 in the basophil autophagy deficiency group was significantly increased, and was positively correlated with the plasma metabolic level and liver function. The present invention includes drugs of OPPC and / or γ-Linolenic acid. The results of drug administration to treat MRL / lpr mice showed 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 OPPC combined with γ-Linolenic acid is significantly better than the therapeutic effect of single administration, which provides a new direction for the clinical treatment of autoimmune liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of plasma biochemical tests on MRL / lpr mice with defective basophil autophagy; in the figure, A is the test result of plasma alanine aminotransferase (ALT) level in MRL / lpr mice, B is the test result of plasma aspartate aminotransferase (AST) in MRL / lpr mice, C is the test result of plasma lactate dehydrogenase (LDH) in MRL / lpr mice, D is the test result of plasma creatine kinase (CK) in MRL / lpr mice, E is the test result of plasma α-hydroxybutyrate dehydrogenase (α-HBDH) in MRL / lpr mice, F is the test result of plasma cholesterol in MRL / lpr mice, G is the test result of plasma high-density lipoprotein in MRL / lpr mice, H is the test result of plasma low-density lipoprotein in MRL / lpr mice, and I is the test result of plasma bile acid in MRL / lpr mice. Figure 3 shows the results of plasma albumin / globulin (A / G) detection in MRL / lpr mice. P < 0.05 indicates a significant difference, which is marked with *.

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

[0021] Figure 3 Figure 3 Correlation analysis between plasma differential metabolites and plasma biochemical indicators in MRL / lpr mice with basophil autophagy defect; in the figure, A is the RDA analysis of differential metabolites and enzymes, and B is the RDA analysis of differential metabolites and proteins and lipids. The length of the arrow indicates 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 indicates the correlation. An acute angle indicates a positive correlation, and an obtuse angle indicates a negative correlation.

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

[0023] Figure 5 The results of H&E staining of liver pathology in MRL / lpr mice treated with OPPC and γ-linolenic acid. DETAILED DESCRIPTION

[0024] 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. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended 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 be obtained from commercial sources.

[0026] Example 1: Plasma biochemical detection of basophil autophagy-deficient MRL / lpr mice

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

[0028] The mice were divided into the following groups:

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

[0030] The plasma of mice in each group was collected, and the biochemical metabolism levels of mouse plasma were detected using a fully automatic biochemical analyzer (Roche, Switzerland). Graphpad Prism 8.0.2 software was used for data statistics and analysis.

[0031] The experimental results are shown in Figure 1 In the plasma enzyme test results, Atg5 - / - The Baso adoptive transfer group was significantly higher than the model control group and Atg5 + / +The plasma ALT, AST and LDH of the Baso adoptive transfer group showed a significant downward trend, reflecting the improvement of liver metabolism; plasma CK showed a downward trend, reflecting the reduction of muscle tissue damage; α-HBDH did not show significant changes. The above results indicate that Baso autophagy deficiency improves the levels of enzymes in the plasma biochemical metabolism of lupus mice.

[0032] In the results of plasma protein and lipid detection, Atg5 - / - The Baso adoptive transfer group was significantly higher than the model control group and Atg5 + / + In the Baso adoptive transfer group, the levels of plasma cholesterol (Cholesterol), bile acid (Bile acid) and low-density lipoprotein (L-density lipoprotein) decreased, while high-density lipoprotein (High-density lipoprotein) did not change significantly; plasma A / G improved. In biochemical metabolism, High-density lipoprotein can transport Cholesterol in the body to various organs of the body, improve immunity, and prevent atherosclerosis. Low-density lipoprotein will cause Cholesterol in the body to further deposit in the blood vessel wall, aggravating the occurrence of atherosclerosis. Bile acid is the end product of Cholesterol metabolism in the liver. In biochemical metabolism detection, the levels of Cholesterol, Bile acid, and Low-density lipoprotein in the plasma of Baso autophagy-deficient lupus mice decreased, while the level of High-densitylipoprotein did not change significantly; plasma Albumin increased, Globulin decreased, and the A / G ratio improved, which shows that Baso autophagy deficiency improves the metabolic level in the body. The above results indicate that Baso autophagy deficiency improves protein and lipid metabolism levels in vivo.

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

[0034] (1) Non-targeted metabolomics analysis of plasma from basophil autophagy-deficient MRL / lpr mice

[0035] The plasma of the basophil autophagy-deficient MRL / lpr mice in Example 1 was sent to Wuhan Maiwei Metabolic Biotechnology Co., Ltd. for plasma non-targeted metabolomics detection.

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

[0037] Results of non-targeted metabolomics in plasma 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 defective group under autoimmune disease state) ( Figure 2 A), Volcano plot analysis showed that 66 metabolites were up-regulated and 33 metabolites were down-regulated in the Atg5KO group ( Figure 2 B).

[0038] The differential analysis of violin metabolites 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 compared with the control group ( Figure 2 C~G).

[0039] Example 3: Disease progression detection in MRL / lpr mice administered with OPPC and γ-linolenic acid

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

[0041] Among them, in the γ-linolenic acid single administration group, mice were orally administered with a vehicle every other day starting from the 8th week, with a dose of 20 mg / kg. In the OPPC single administration group, mice were orally administered with a vehicle every other day starting from the 8th week, with a dose of 20 mg / kg. In the OPPC combined with γ-linolenic acid administration group, γ-linolenic acid and OPPC were mixed in equal amounts, 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) The plasma biochemical indices (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 a fully automatic biochemical analyzer. The detection method of MRL / lpr mouse plasma samples was the same as that in Example 1.

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

[0044] Dewaxing: Place the liver tissue slides in xylene I for 20 min and in xylene II for 20 min; Place the slides in anhydrous ethanol I for 5 s, anhydrous ethanol II for 5 s → 95% alcohol I for 5 s, 95% alcohol II for 5 s → Wash with water for 15 s; Place the slides in Subenin staining solution for 2 min → Wash with water for 15 s×2 → 1% hydrochloric acid alcohol for 3 s → Wash with water for 15 s → 1% ammonia water for 3 s → Wash with water for 15 s; Place the slides in eosin staining solution for 2 min → Wash with water for 15 s → anhydrous ethanol for 2 s → Bake dry, seal with neutral resin; Take pictures.

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

[0046] In summary, the plasma biochemical metabolism level and liver pathology of the γ-Linolenic acid single administration group and the OPPC single administration group were improved compared with the Control group, indicating that γ-Linolenic acid or OPPC has an improving effect on disease progression. However, it is worth noting that the OPPC combined with γ-Linolenic acid administration group had a more obvious improvement in disease progression compared with the γ-Linolenic acid single administration group and the OPPC single administration group, indicating that the treatment effect of OPPC combined with γ-Linolenic acid is significantly better than that of a single treatment. The above results show that OPPC combined with γ-Linolenic acid treatment can significantly improve the disease progression of 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 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. A drug for treating autoimmune liver disease, characterized in that: It includes 1-Oleoyl-2-palmitoy-sn-glycero-3-phosphocholine (OPPC) and / or γ-linolenic acid, or derivatives of OPPC and / or γ-linolenic acid.

2. The drug according to claim 1, characterized in that It also includes pharmaceutically acceptable carriers or excipients.

3. The drug according to claim 1, characterized in that The medicine is an oral preparation or an injection preparation.

4. Use of OPPC and / or γ-Linolenic acid in the preparation of drugs for preventing and / or treating autoimmune liver diseases.

5. The use according to claim 4, characterized in that: When the drug is administered, the dosage of OPPC and / or γ-linolenic acid is 10 mg / kg to 20 mg / kg once, once every 1 to 2 days.

6. The use according to claim 4, characterized in that: The medicine is an oral preparation or an injection preparation.

7. The use according to claim 4, characterized in that: The function of the drug is at least one of the following: Ⅰ. Reduce the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, cholesterol and low-density lipoprotein in plasma; Ⅱ. Increase the level of high-density lipoprotein and A / G ratio in plasma; III. Improve the level of liver cell degeneration and necrosis.

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