The Protective Effect And Mechanism Of Betulinic Acid On Liver Injury In Cyclophosphamide-induced Mice

Background: Betulinic acid(BA),a pentacyclic lupine-type triterpene compound,is mainly distributed in birches(Betulaceae),and exhibits many biological effects including anti-inflammatory,anti-oxidant,anti-tumor and anti-virus.Previous study has found that BA has a protective effect on alcoholic liver injury by inhibiting mitogen-activated protein kinase(MAPK)and endoplasmic reticulum stress(ERs)signaling pathways.Besides,BA attenuates intestinal injury induced by cyclophosphamide(CYP)via the inhibition of nuclear factor kappa-B(NF-κB)/MAPK signaling pathway and the activation of nuclear factor erythroid-2related factor 2(Nrf2)signaling pathway.However,little is known about the protective effects and potential mechanisms of BA on CYP-induced liver injury.Objective: The aim of this study was to investigate the protective effects and potential protective mechanisms of BA on CYP-induced liver injury in mice which may provide theoretical support for BA as a feed additive and its further clinical application.Methods:(1)After adaptive feeding for one week,60 Kunming mice were divided into 6 groups(n=10): control group,CYP group,BA(0.5 mg/kg)group,BA low,medium and high dosages(0.25,0.5,1.0 mg/kg)and CYP co-treatment groups.The mice were gavaged with different dosages of BA for 14 days,and then intraperitoneal injection of 100 mg/kg CYP on 14 th and 15 th days to induce liver injury model.Serum and liver were collected for the following experiments:The protective effect of BA on CYP-induced liver damage in mice: The serum levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)were detected by the automated blood analyzer.H&E staining was performed to observe histological changes,and the Tunel method was performed to measure the cell apoptosis in liver.The superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)glutathione(GSH)and malondialdehyde(MDA)levels of liver were detected by reagent kits,and reactive oxygen species(ROS)content was detected by immunofluorescence.The m RNA expression of antioxidant enzymes SOD,GSH-Px and CAT,as well as the inflammatory cytokines including interleukin-1beta(IL-1β),IL-6,IL-10 and tumor necrosis factor α(TNF-α)were determinated by q PCR.The effect of BA on nuclear transcription factor kappa B(NF-κB)/mitogen-activated protein kinase(MAPK)/ nuclear factor E2-related factor 2(Nrf2)and mitochondrial signaling pathways in liver injury caused by CYP: The protein expressions and phosphorylation of p38,c-jun N-terminal kinase(JNK)and extracellular signal-regulated kinase(ERK)in MAPK signaling pathway,and NF-κB and NF-κB inhibitor-α(IκBα)in NF-κB signaling pathway were determinated by Western blot.Meanwhile,the protein expressions of Nrf2 and heme oxygenase-1(HO-1)in Nrf2 signaling pathway,and B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and caspase-9(Caspase-9)in mitochondrial signaling pathway were detected by Western blot.The m RNA expression of dynamin-related protein1(Drp1),mitochondrial fission factor(Mff),mitochondrial fission protein 1(FIS1)and optic atrophy 1(OPA-1)were measured by q PCR.(2)After adaptive feeding for one week,50 Kunming mice were divided into 5groups(n=10): control group,CYP group,BA(0.5 mg/kg)+CYP group,PD98059(ERK inhibitor)+CYP group and BA(0.5 mg/kg)+PD98059+CYP group.The mice were orally administrated with BA once a day and injected intraperitoneally with PD98059 once two days for 14 days,respectively.And then intraperitoneal injection of 100 mg/kg CYP on 14 th and 15 th days to induce liver injury model.Serum and liver were collected for detection.The AST and ALT levels of serum were detected by the automated blood analyzer.H&E staining and transmission electron microscope were used to observe histological changes and ultrastructure changes of liver cells,respectively.The liver cell apoptosis was detected by Tunel method.The protein expression and phosphorylation of JNK and ERK,and protein expression of Bcl-2,Bax and Caspase-9 were measured by Western blot.Results:(1)BA pretreatment alleviated vacuole-like degeneration,loose of cytoplasm,lightly stained and enlarged of nuclei in hepatocytes caused by CYP,and decreased the serum level of AST and ALT.BA upregulated the m RNA expression of GSH-Px,CAT,GSH,down-regulated the activity of SOD compensatorily,and cleared the ROS accumulation in liver.Besides,BA reduced hepatocyte apoptosis caused by CYP.What’s more,BA pretreatment inhibited the m RNA expressions of IL-1β and TNF-α,promoted the m RNA expression of IL-10,as well as promoted the m RNA expression of IL-6 compensatorily.These results indicated that BA had a protective effect on CYP-induced liver injury by inhibiting the changes of the serum enzyme caused by CYP,alleviating the lipid peroxidation,inhibiting the secretion of pro-inflammatory cytokines and promoting the secretion of anti-inflammatory cytokine.(2)BA pretreatment downregulated the ratios of p-ERK/ERK and p-JNK/JNK,and upregulated the ratio of p-p38/p38,p-NF-κB/NF-κB and p-IκBα/IκBα in MAPK/NF-κB signaling pathways.Besides,BA activated the protein expression of Nrf2 and HO-1 in Nrf2 signaling pathway.Furthermore,BA regulated the division and fusion of mitochondria by inhibiting the m RNA expressions of Drp1,Mff,FIS1 and activiating m RNA exoression of OPA1.At the same time,BA alleviated hepatocyte apoptosis by increasing the ratio of Bcl-2/Bax,and decreasing the ratio of Cleave-Caspase-9/Pro-Caspase-9.These results indicated that BA had a protective effect on CYP-induced liver damage by regulating the MAPK/NF-κB/Nrf2 and mitochondrial signaling pathways.(3)Pretreatment with BA and PD98059 restored the structure of the liver sinusoids,reduced vacuolation and restored the mitochondrial morphology in liver cell.BA and /or PD98059 reduced the increase of AST and ALT serum levels induced by CYP.BA and/or PD98059 decreased the hepatocyte apoptosis caused by CYP.Besides,BA and/or PD98059 pretreatment inhibited the ratios of p-ERK/ERK and Cleave-Caspase-9/ Pro-Caspase-9,and promoted the ratio of Bcl-2/Bax.The results demonstrated that BA prevented CYP-induced liver damage by inhibiting ERK-mediated mitochondrial signaling pathway.Conclusion: BA shown protective effects on CYP-induced liver damage by alleviating inflammation and antioxidative stress and restoring the balance of mitochondrial fusion and division throught partly inhibition of ERK-mediated mitochondrial signaling pathway.