Effects Of Salvia Miltiorrhiza Polysaccharides On TLR4/MyD88 Signaling Pathway And Apoptosis Factors In Liver Of Immunological Liver Injury Mice

A good liver physiological state is an important prerequisite for maintaining animal health.A variety of harmful factors can lead to liver injury.Once the liver is damaged,it will cause great harm to the body and even endanger the life of the animal.In recent years,the incidence of animal liver diseases in the aquaculture industry has increased significantly due to the intensive farming model and the abuse of antibiotics,especially in the case of immunological liver injury.In addition,the mechanism of animal immunological liver injury is very complicated and has not been thoroughly studied.Therefore,it is extremely important to study the mechanism of animal immunological liver injury and the drugs that alleviate animal immunological liver injury.Studies have shown that Salvia miltiorrhiza polysaccharides have a certain protective effect on liver injury.However,the specific mechanism of Salvia miltiorrhiza polysaccharides to alleviate immunological liver injury is not yet fully understood.Therefore,in order to explore the possible mechanism of animal immunological liver injury and provid experimental basis for the development of Salvia miltiorrhiza polysaccharides as a new Chinese medicine for the prevention and treatment of animal immunological liver injury in the future,this study focused on the regulation of Salvia miltiorrhiza polysaccharides on TLR4/MyD88 signaling pathway and apoptotic factors in liver of immunological liver injury mice induced by LPS combined with D-GalN.120 Kunming mice were randomly divided into the normal control group,model group,positive drug(bifendate 200 mg/kg)control group and low,medium and high dose of Salvia miltiorrhiza polysaccharides(250 mg/kg,500 mg/kg,750 mg/kg)protection group.Each group of mice were given the same amount of normal saline or the corresponding drugs by gavage daily for 12 consecutive days.After the last dose for 1 hour,except for the normal control group,mice in the other groups were intraperitoneally injected with LPS(10 mg/kg)and D-GalN(300 mg/kg)to establish a model of immunological liver injury in mice.After 3 hours of modeling,blood samples were obtained by retro-orbital blood collection.Subsequently,these mice were sacrificed by cervical dislocation under full anesthesia,and the liver tissues were dissected immediately.HE staining was used to observe the pathological changes of liver tissue in each group.Detecting the levels of MDA,SOD and GSH-PX in the liver tissue in mice of each group.The concentration of IL-1β and TNF-α in the serum and liver tissue of experimental mice were detected by ELISA.The expression levels of CXCL-10 and ICAM-1 in liver tissue were detected by Immunohistochemistry.The mRNA relative expression levels of LBP,CD14,MD-2,TLR4 and MyD88 in liver tissue were detected by qRT-PCR.The protein expression levels of TLR4,MyD88,P-IKK-α/β,P-IκB-α and P-NF-κB(P-P65)were detected by Western blot.The expression levels of Bax,Bcl-2,Fas,Fas-L and Caspase-3(P17)were detected by Western blot and immunohistochemistry.The results showed that compared with the normal control group,hepatocytes of model group were swollen and there were a lot of inflammatory cell infiltration and patchy necrosis in the liver parenchyma in mice of model group.The concentration of MDA in the liver tissue was increased significantly(P<0.01),the activity of SOD and GSH-PX were decreased significantly(P<0.01).The concentration of IL-1β and TNF-α in the serum and liver tissue were increased significantly(P<0.01).The expression levels of CXCL-10 and ICAM-1,the mRNA levels of LBP,CD14,MD-2,TLR4 and MyD88,the protein levels of TLR4,MyD88,P-IKK-α/β,P-IκB-α and P-P65 were all increased significantly(P<0.01).The expression levels of Bax,Fas-L and Caspase-3(P17)were significantly increased(P<0.01),the level of Bcl-2 was significantly decreased(P<0.01)and the difference in the level of Fas is not significant(P>0.05).Compared with model group,the hepatocytes of the Salvia miltiorrhiza polysaccharides groups and the positive drug control group tend to be intact,and the number of inflammatory cells and necrosis in the liver parenchyma were reduced in mice of the Salvia miltiorrhiza polysaccharides groups and the positive drug control group.The concentration of MDA in the liver tissue was decreased significantly(P<0.01),the activity of SOD and GSH-PX were increased significantly(P<0.05 or P<0.01).The concentration of IL-1β and TNF-α in the serum and liver tissue were decreased significantly(P<0.05 or P<0.01).The expression levels of CXCL-10 and ICAM-1,the mRNA levels of LBP,CD14,MD-2,TLR4 and MyD88,the protein levels of TLR4,MyD88,P-IKK-α/β,P-IκB-α and P-P65 were all decreased significantly(P<0.05 or P<0.01).The expression levels of Bax,Fas-L and Caspase-3(P17)were significantly decreased(P<0.05 or P<0.01),the level of Bcl-2 was significantly increased(P<0.01)and the difference in the level of Fas is not significant(P>0.05).The medium dose of Salvia miltiorrhiza polysaccharides was most effective in regulating each factor(P<0.05 or P<0.01).Conclusion: This study demonstrates that LPS combined with D-GalN can induce excessive oxidative,inflammatory and apoptotic reactions in the liver of mice,and ultimately induce immunological liver injury.It was also preliminarily confirmed that Salvia miltiorrhiza polysaccharides could effectively regulate TLR4/MyD88 signaling pathway and apoptotic factors in liver of mice with immunological liver injury induced by LPS combined with D-GalN,inhibit excessive oxidative,inflammatory and apoptotic reactions in liver,and then alleviate immunological liver injury.