Mechanism Of The Protective Effect Of Kaempferol On Acetaminophen-induced Hepatotoxicity Injury

Objective:Based on NLRP3 and HMGB1/TLR/NF-signaling pathways,the effects of kaempferol on acetaminophen-induced hepatotoxicity and related mechanisms were studied.Methods:⑴ Replicated the model of acute liver injury induced by APAP: C57BL/6 mice were randomly divided into 4 groups,normal control group(control group),the model group of acetaminophen-induced liver injury(APAP group),low dose of kaempferol(30mg/kg)group(APAP+KA30 group),high dose of kaempferol(60mg/kg)group(APAP+KA60 group),Ten in each group.normal control group(control group)and the model group of acetaminophen-induced liver injury(APAP group)Gavage appropriate amount of normal saline according to body weight,once daily.low dose of kaempferol(30mg/kg)group(APAP+KA30 group)and high dose of kaempferol(60mg/kg)group(APAP+KA60 group):An appropriate amount of kaempferol suspension(30,60mg/kg)was administered respectively according to body weight,once daily.Mice were given continuous gavage for 7 days.On the 8th day,paracetamol solution was intraperitoneally injected into each group except the Control group,the dose is 300mg/Kg.Blood and liver tissue samples were collected 24 hours after intraperitoneal ⑵injection.ALT and AST in serum was determined by special kit.⑶ A sample of the liver tissue was collected,processed and H&E stained to be examined under an optical microscope for possible tissue damage.Immunohistochemical staining of HMGB1 and TLR4 was performed.⑷ SOD,GSH and MDA in liver tissue was determined by special kit.⑸ Western blot was used to detect the expression of HO-1 /NQO1 pathway protein in the liver tissues of mice in each group.⑹ Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL)staining was used to assay cellular apoptosis,and Western blot was used to detect the expression of apoptosis-related proteins(bcl-2 and bax)in the liver tissues of mice in each group.⑺ The m RNA expression of il-1 expression,il-6 expression and TNF-α expression in the liver tissues of each group was determined by qPCR.⑻ Protein western blot was used to detect the expression of HMGB1/TLR4/NF-κβ pathway related proteins in the liver tissues of mice in each group.⑼ Protein western blot was used to detect NLRP3 protein expression in liver tissues of mice in each group.Results:(1)kaempferol can inhibit the elevation of AST and ALT in serum caused by acute liver injury induced by APAP.(2)kaempferol can reduce the pathological changes of liver tissue caused by APAP-induced acute liver injury.(3)kaempferol can inhibit the decrease of total SOD and GSH level induced by acute liver injury induced by APAP,and simultaneously inhibit the increase of MDA level.(4)kaempferol can reduce the apoptosis of liver cells caused by acute liver injury induced by APAP,inhibit the decrease of bcl-2 protein expression level,and inhibit the increase of bax protein expression level.(5)the expression of HMGB1/TLR4/NF-κβ pathway protein could be down-regulated by kaempferol.(6)kaempferol can up-regulate the protein expression of HO-1/NQO1 pathway.(7)kaempferol down-regulated the expression of NLRP3 protein.(8)kaempferol can inhibit the expression of inflammatory cytokines IL-1β,IL-6 and TNF-α.Conclusion: kaempferol has a protective effect on APAP-induced acute liver injury,the specific mechanism may be related to kaempferol to reduce the inflammatory response by inhibiting the expression of IL-1β,IL-6 and TNF-α,and down-regulating the expression of NLRP3 protein.Oxidative stress and oxidative damage were reduced by up-regulating HO-1 /NQO1 pathway protein expression,increasing total SOD and GSH,and decreasing MDA.The apoptosis was reduced by increasing the expression of bcl-2 protein and inhibiting the expression of bax protein.The expression of HMGB1/TLR4/NF-κβ pathway was down-regulated to reduce apap-induced acute liver injury.