Study On MSCs Treatment Of AIH By Change The Polarization State Of Macrophages In Mice

Purpose:Autoimmune hepatitis is a chronic progressive liver inflammatory disease mediated by autoimmunity.Studies have confirmed that mesenchymal stem cells have achieved certain clinical effects in the treatment of AIH,but the specific mechanism is still controversial.More and more studies have shown that macrophages play an important role in MSC repair and damage to the liver,but the mechanism by which macrophages affect mesenchymal stem cells to repair damaged liver is still unclear.It is widely believed that inflammation is associated with oxidative stress,and Nrf2 is a key factor in the body's defense against oxidative stress,which promotes HO-1 transcription,and HO-1 regulates and participates in the body's anti-inflammatory,anti-apoptotic and anti-oxidative stress is universally expressed in macrophages.Studies have shown that HO-1 stimulates M1 macrophages to transform M2 macrophages.In this study,transplanted mesenchymal stem cells were used to treat autoimmune hepatitis in mice,and M1 macrophages in liver after mesenchymal stem cell transplantation were explored.Whether the number of cells M1 and M2 type macrophages changes is related to HO-1.To investigate whether the polarization of macrophages and the therapeutic effect of mesenchymal stem cells are related HO-1,and provide a theoretical basis for the future treatment of autoimmune hepatitis by mesenchymal stem cells.METHODS:Part I: Mouse bone marrow mesenchymal stem cell transplantation for autoimmune hepatitis mice1.Establishment of an acute AIH mouse model: Female mice were injected with ConA to construct an acute autoimmune hepatitis model.After the model was established,serum alanine aminotransferase,aspartate aminotransferase and liver histopathological results were used to determine whether the model was successful.2.Efficacy evaluation of mouse bone marrow mesenchymal stem cells: After AIH mice were modeled,they were divided into AIH+MSCs group and AIH group.AIH model mice were injected with mesenchymal stem cells through the tail vein,andcontrol AIH mice.An equal volume of PBS was injected via a rat tail,and an equal volume of PBS was injected into the normal group.The liver function,liver pathology,hepatocyte apoptosis and proliferation of the three groups of mice were compared.The expression of TNF-? IFN-? and IL-6 in the supernatant of mice was detected by ELISA.TNF-in liver tissue was detected by RT-PCR.The expression of ? IFN-? and IL-6 was evaluated to evaluate the effect of mesenchymal stem cells on acute autoimmune hepatitis mice.Part II: Mechanism of macrophage polarization in the treatment of mouse bone marrow mesenchymal stem cells1.Distribution of M1/M2 in intrahepatic macrophages in mice: It was divided into AIH+MSCs+Znpp group and AIH+Hemin group by HO-1 inhibitor Znpp and agonist Hemin.Immunofluorescence double staining was used to detect the distribution of M1 macrophages and M2 macrophages in the liver tissues of AIH group,AIH+MSCs group,AIH+MSCs+Znpp group and AIH+Hemin group.RT-PCR was used to detect the expression of INOS and Arg1 in mRNA to reflect the distribution of M1macrophage(INOS)and M2 macrophage(Arg1)in liver tissue.2.Molecular pathway of macrophage polarization: In the normal group,AIH group,AIH+MSCs group,AIH+MSCs+Znpp group and AIH+Hemin group,the expression of HO-1 and Nrf2 in liver tissues of each group was detected by Western and RT-PCR.The pathological changes of the visceral tissues,the apoptosis and proliferation of liver tissues,and the expression of TNF-? IFN-? and IL-6 in the supernatant and liver tissues of the mice were detected.RESULTS:Part I: Experimental study of mesenchymal stem cell transplantation in the treatment of autoimmune hepatitis mice.1.Evaluation of autoimmune hepatitis model: Compared with the normal group,serum biochemical parameters of alanine aminotransferase and aspartate aminotransferase were significantly increased in the AIH group,and the difference was statistically significant(P<0.05).Liver pathology shows that inflammatory cells infiltrate liver tissue,a large number of liver cell necrosis,intrahepatic vasodilation,mesenchymal hyperemia,hepatic sinusoidal disorder,nuclear pyknosis,nuclearfragmentation,nuclear dissolution,in line with AIH model standards,modeling established.2.Efficacy evaluation: Compared with AIH group,alanine aminotransferase and aspartate aminotransferase decreased in AIH mice treated with MSCs;liver cell necrosis,mesenchymal hyperemia,hepatic sinusoidal disorder,nuclear pyknosis and nucleus were observed in liver pathology.The fragmentation and nuclear dissolution were improved;the hepatocyte apoptosis was significantly improved;the hepatocyte proliferation was obvious;the expression of TNF-? IFN-? and IL-6 was decreased,and the above indexes were statistically significant(P<0.05).Part II: Study on the mechanism of macrophage polarization1.Compared with AIH group,M2 macrophages were the main cells in MSCs group,while M1 macrophages were the main AIH group,the difference was statistically significant(P<0.05).2.Molecular pathway of macrophage polarization: Compared with AIH group and AIH+MSCs+Znpp group,liver necrosis,nuclear pyknosis,nuclear fragmentation and nuclear dissolution were significantly improved in AIH mice treated with MSCs and Hemin;Nrf2 and The expression of HO-1 was increased.The expression of M2 macrophage was increased,while the expression of M1 macrophage was inhibited;the apoptosis of liver cells was significantly improved,and the proliferation of hepatocytes was significantly increased(P<0.05).Proinflammatory TNF-The expression of ? IFN-? and IL-6 was decreased,and the difference was statistically significant(P<0.05).Conclusion:1.ConA-induced autoimmune hepatitis mouse model can mimic the pathology,liver damage and inflammation of patients with autoimmune hepatitis.Mouse bone marrow mesenchymal stem cells improve liver function and damaged liver tissue in mice with autoimmune hepatitis,reduce hepatocyte apoptosis,increase hepatocyte proliferation,and inhibit the expression of pro-inflammatory factors.2.MSCs promote the polarization of M1 macrophages to M2 macrophages in liver tissue through Nrf2/HO-1 signaling pathway,and maintain the stability of liver inflammation in the mouse model of autoimmune hepatitis,and play a role inprotecting liver tissue.