Protectives Effects Of Kaempferol Against Acetaminophen-induced Acute Liver Injury In Mouse

Objective: This experiment aims to explore the effect of kaempferol on AILI(acetaminophen-induced acute liver injury)and its mechanism.To develop kaempferol become a drug for acute liver injury,which provide pharmacological basic research theories.Methods: 1.LO2 cells experiment,grouping and model replication.LO2 cells APAP acute liver injury model: logarithmic growth cells were divided into blank control group,model group,kaempferol low dose group(5?M),kaempferol Medium dose group(10?M),kaempferol high dose group(20?M).Except for the blank control group,the other groups were treated with 5 m M APAP for 24 hours.Kaempferol treatment groups were treated with different concentrations of kaempferol.The cytological changes of each group and the ratio of LDH released by the cells were observed.The mechanism of the signal protein p-JNK and Endo G was used to detect acute liver injury induced by kaempferol-resistant APAP.2.Mouse primary hepatocyte experiment,grouping and model replication.The mouse liver cells were extracted from female mice.After the primary hepatocytes adhered to the wall,the primary hepatocytes were divided into blank control group and model group.The low dose of kaempferol,medium dose of kaempferol,high dose of kaempferol,except for the blank control group,all groups were treated with 10 m M APA for 12 hours,The kaempferol-treated group was treated with different concentrations of kaempferol at the same time,and then the LDH released ratio and cell GSH were measured 3.Experimental animals,grouping and model replication.SPF male Balb/c mice were randomly divided into 3 groups: blank control group,APAP model group and kaempferol administration group,according to the difference after APAP administration.At the time point,each group was divided into 3 groups of 1.5 hours,3 hours and 24 hours.Each group have 7 mice.Except the blank control group,the other groups were fasted for 22 hours to induce acute APAP injury model.(19 : 00-the next day 17:00),250mg/kg intraperitoneal injection,injection volume: 20?L/g.kaempferol dose group kaempferol dose 80mg/kg,2 hours before APAP intraperitoneal injection.At 1.5 hours,3 hours,and 24 hours,and the blood was taken for further experiments.The mice were sacrificed and the liver tissue was removed into two parts.Some of the dehydrated cells were used for paraffin sectioning,and the left were frozend for subsequent use.Molecular experiments such as protein and RNA extraction and other experiments.The detection of serological markers,histopathological observation,and the quantitative detection of acute liver injury by IHC,and the q PCR were used to determine the therapeutic effect of kaempferol on AILI.Results: 1.Compared with the APAP group,the morphology of LO2 cells in the KA treatment group was significantly improved,the apoptotic cells were significantly decreased,the LDH release rate was significantly decreased,and the Western Blot results showed that the treatment was performed by kaempferol.Afterwards,the expression of p-JNK and Endo G protein in hepatocytes was significantly down-regulated.2 Compared with the APAP group,the release rate of LDH in the primary hepatocytes of the kaempferol treatment group was significantly decreased,and the GSH content was significantly up-regulated.3 Compared with the APAP group,HE staining showed a significant decrease in liver necrosis area in the kaempferol treatment group,and liver GSH increased significantly Serum ALT decreased significantly,and the survival status was significantly improved.TUNEL results showed that compared with the APAP group,the TUNEL positive staining of the kaempferol group was significantly reduced;the liver signal protein(p-JNK,JNK,Endo G,Bcl-2)were detected by western blot.Compared with APAP group,the contents of p-JNK and Endo G protein in kaempferol treatment group were significantly down-regulated,and the contents of Bcl-2 protein was significantly up-regulated.Real-time PCR was used to detect ARE-related genes and their downstream genes(Nrf2,Gcl-c,Sod2,Ugt1a1,Ugt1a9,Sult1a1,GST?,Mrp2,Mrp4).The results showed that compared with the APAP group,Nrf2,Gcl-c,Sod2,Ugt1a1,Ugt1a9,Sult1a1,GST? and Mrp2 were significantly up-regulated;Mouse liver macrophage marker protein CD68 and neutrophil marker protein Ly6 G were detected by immunohistochemistry,compared with APAP group,the positive staining area of CD68 and Ly6 G in KA group decreased significantly;The related pro-inflammatory factors and chemokines showed that IL-1B,IL-6,TNFa were significantly down-regulated in the KA-treated group compared with the APAP group,but IL10 was no significantly different,ccl2,ccl3,ccl4 and ccl7 were significantly down-regulated.Conclusion: Kaempferol has the effect of protecting APAP-induced acute liver injury,and its mechanism may be anti-oxidative stress and inhibition of inflammatory response.