Effect Of Pterostilbene On Inhibiting Vascular Endothelial Immunoproteasome Mediated Antigen Presentation And Improving Nonalcoholic Steatohepatitis

Objective:Small chemical molecules of Chinese medicine are the active components in Chinese herbal medicine.Modern pharmacology has proved that they have many medicinal properties,such as anti-oxidation,anti-cancer,anti-bacteria,and so on,which are important sources of new drug development.Through literature review in recent years,we have found that Pterostilbene（PT）is a polyhydroxystilbene compound and an important antifungal active ingredient in Pterocarpus tatarinowii.PT has a positive protective effect on the liver,but the specific mechanism is not clear,and the related research literature is scarce.Chronic or severe liver injury with fibrosis is one of the main histological features of nonalcoholic steatohepatitis（NASH）and is also an important cause of liver cirrhosis.Vascular endothelial cells play an important regulatory role in the process of hepatic fibrosis.The formation of MHC-I and MHC-II is a prerequisite for the activation of various T cells.In NASH,the massive production of MHC-I and MHC-II complexes will over-activate immune cells and lead to the release of a large number of inflammatory factors to aggravate liver fibrosis.On this basis,we mainly studied whether PT could inhibit the antigen presentation of liver endothelial cells MHC-I and MHC-II by inhibiting the activity of immune proteasome,and improve liver fibrosis to reduce the development of NASH,and reveal its potential mechanism,thereby providing new ideas for the development of new drugs for the treatment of NASH.Methods:A:Nonalcoholic steatohepatitis（NASH）model and drug therapy:The 9-week-old mice were first selected and randomly divided into a control group and a steatohepatitis group.Patients in the control group were given ordinary feed and drank water every day for 12 weeks/24 weeks.The non-alcoholic steatohepatitis model group was given a high-fat diet（21.1%fat+41%sucrose+1.25%cholesterol）and high-glucose solution（23.1 g/L d-fructose+18.9g/L d-glucose）daily for 12 days,and intraperitoneal injection of 20%CCl₄solution every three days.PT 10 mg/kg/d（diluted with normal saline）was administered intraperitoneally on the second day after injection of CCl₄,and samples were taken on the second day after the last dose（Day 12）.The experimental mice were euthanized with pentobarbital injection and dissected separately,and the liver tissues and serum samples were collected.The liver function of the mice was evaluated by ALT and AST kits.Pathological staining techniques of HE and Masson were used to evaluate the liver tissue damage.Immunohistochemistry was used to assess the expression of fibrosis indicatorsα-SMA and Collagen I and the degree of fibrosis.IL-1β,IL-6 and IL-18 were detected by immunofluorescence staining.Immune proteasome LMP7;Expression of MHC-I and MHC-II proteins;The gene levels of IL-1β,IL-6 and IL-18 were detected by real-time PCR.B:In vitro cell model and drug treatment:After the EOMAs cells grew to an appropriate density,mouse recombinant IFN-γ（100ng/ml）was used to stimulate and induce the expression of MHC-I and MHC-II in EOMAs cells,followed by simultaneous intervention with different concentrations of PT（10μM,2μM,0.4μM,0.08μM）.After 24 hours of drug intervention,RNA was collected from the cells for real-time PCR experiment detect the changes of MHC-I and MHC-II m RNA levels.Results:（1）PT improves the liver physiological function of NASH model mice and relieves liver fibrosis.Serum ALT and AST were significantly down-regulated in the PT administration group（P<0.05）.H&E staining and MASSON staining showed that the hepatic fibrosis area of NASH mice after PT treatment was decreased（P value<0.01）.Immunohistochemical experiments showed thatα-SMA,Collagen-I,and other fibrosis indicators were decreased significantly after PT administration（P value<0.01）.（2）PT improves the inflammatory response in NASH model mice.The expression levels of IL-1β,IL-6,and IL-18 in hepatocytes of rats after PT administration were significantly decreased（P<0.01）.Meanwhile,the m RNA levels of IL-1β,IL-6,and IL-18 in hepatocytes also showed a significant downward trend after PT administration（P<0.05）.（3）PT reduces the abnormal activation of CD4⁺T and CD8⁺T cells by inhibiting the activity of the immunoproteasome LMP7 and thereby reducing the expression of vascular endothelial MHC-I and MHC-II.Compared with the model group,after PT intervention,the expression levels of endothelial MHC-I and MHC-II were decreased,and the number of CD4⁺T and CD8⁺T in hepatocytes was significantly decreased（P value<0.01）.（4）Delete CD4⁺T cells to alleviate liver fibrosis caused by chronic liver injury.The results of pathological staining showed that compared with the model group of hepatic fibrosis,the liver tissue of the CD4⁺T deletion group was improved and the degree of hepatic fibrosis was significantly decreased（P<0.01）.（5）The expression of proteasome-associated antigen-presenting cells in endothelial cells of patients with liver cirrhosis increased.Immunostaining showed that compared with non-cirrhotic patients,MHC-II expressed in the liver of cirrhotic patients was mostly co-located with CD31,and some with LYVE-1（labeled lymphatic endothelium）.At the same time,a large amount of proteasome LMP7 is also expressed in the liver of patients with liver cirrhosis,and co-located with CD31.Conclusion:PT reduced the expression of MHC-I and MHC-II in vascular endothelial cells and their antigen presentation to CD4⁺and CD8⁺T cells by inhibiting the activity of immunoproteasome LMP7,thereby reducing the intrahepatic inflammatory response and improving liver fibrosis in NASH mice.