| Objective:This study was intended to investigate whether the Acanthus ilicifolius alkaloid A(HBOA)can alleviate the injurious effect of high-fat diet(HFD)induced non-alcoholic fatty liver disease(NAFLD)in rats.Furthermore,it was also further investigated whether HBOA exerts its mechanism of action against NAFLD by regulating the expression of hepatic lipogenesis-related factors and endoplasmic reticulum stress(ERS)signaling pathway genes and proteins in NAFLD rats.Method:Sixty Sprague Dawley male rats were randomly divided into two groups:group I rats(10 animals)fed with a basic diet and group II rats(50 animals)fed with a high-fat diet to replicate the NAFLD model.After 8 weeks of continuous feeding,2 rats in group I and 5 rats in group II were randomly selected for liver pathological analysis according to the gold standard for NAFLD model judgment.After confirming the success of NAFLD modeling in rats,the rats in group II were randomly divided into the model group,HBOA high-dose group,HBOA mediumdose group,HBOA low-dose group,Silibinin group,and the rats in group I were used as the normal control group,with 8 rats in each group.From 9 weeks onwards,in addition to continuing to give a high-fat diet,rats in the Silibinin group were intragastrically administered with Silibinin 26.25 mg/kg/d,and rats in the low,medium and high-dose HBOA groups were intragastrically administered with HBOA 25,50 and 100 mg/kg/d,respectively,while rats in the normal control group and the model group were intragastrically administrated with an equal volume of 0.6% CMC-Na until the end of 12 weeks.The rats were deprived of food except for water for 10 hours.The serum and liver samples were collected and stored the next morning for subsequent detection and analysis.The levels of AST,ALT,TP,ALB,TG,TC,HDL-C and LDL-C in the serum were determined using an automatic biochemical analyzer,and the FFA content was detected by the kit.The contents of SOD,MDA,GSH,GSH-Px and MPO in the liver were detected by the kit.In addition,liver sections were prepared to evaluate the pathological changes and collagen deposition in the liver by H&E staining and Masson’s trichrome staining.The hepatocyte lipid droplet deposition and apoptosis were examined by saturated oil red O staining and TUNEL staining.The expressions of PERK,EIF2α,ATF6,GRP78 and CHOP in liver tissues were observed by immunohistochemistry.Moreover,RT-PCR and Western blot methods were used to detect the genes and proteins expression of the endoplasmic reticulum stress signaling pathway and lipid synthesis-related factors,as well as the expression of inflammation and apoptosis-related proteins.Results:1.HBOA had a protective effect on liver injury in NAFLD ratsThe results showed that the activities of AST and ALT in the serum of the model group were significantly increased,and the A/G ratio was inverted;after HBOA treatment,the activities of AST and ALT in serum were significantly reduced and the A/G ratio was restored.Furthermore,H&E and Masson’s staining demonstrated that there was no significant steatosis in hepatocytes of a normal control group,and the structural sequence was clear and complete,there was no lesion area,and only a small amount of collagen was deposited on the vascular wall;The hepatocytes of the model group showed extensive steatosis and fat vacuoles,loose cytoplasm and disordered structure,a little eosinophilic necrosis and inflammatory cell infiltration in the portal area,and significantly increased collagen deposition;however,the HBOA treatment group could significantly decrease the degree of liver lesions.These results suggested that HBOA could ameliorate the degree of liver lesions by alleviating hepatocyte damage and collagen deposition and reducing serum enzyme activity in NAFLD rats.2.HBOA alleviated oxidative stress and lipid peroxidation in NAFLD ratsCompared with the normal control group,the activities of GSH-Px,SOD and GSH in the liver tissue of the model group were significantly decreased,and the content of MDA was significantly increased.Whereas,after intervention by HBOA,the abnormal changes of these levels could be significantly reversed.These results indicated that the therapeutic effect of HBOA on NAFLD rats may be related to its inhibition of oxidative stress,the reduction of lipid peroxidation and enhancement of antioxidant enzyme activity.3.HBOA attenuated inflammation response in NAFLD ratsCompared with the normal control group,the MPO content in the liver tissue of the model group was markedly increased,and it was markedly decreased after HBOA treatment.Western blot results also demonstrated that compared with the normal control group,the phosphorylation levels of NF-κBp65 and IκBα proteins in the liver tissues of the model group were notably up-regulated.But after HBOA treatment,it can notably down-regulated the phosphorylation levels of NF-κBp65and IκBα proteins in liver tissues.These results indicated that HBOA could affect the activation of key factors in the NF-κB signaling pathway caused by HFD,thereby reducing cell inflammatory response.4.HBOA alleviated hepatocyte apoptosis in NAFLD ratsThe results of TUNEL staining showed that compared with the normal control group,there were a large number of apoptotic cells in the liver tissue of the model group;Compared with the model group,the number of apoptotic cells in the HBOA intervention group was reduced to a certain extent.Moreover,the western blot results also confirmed that the expression of pro-apoptotic proteins Bax,Caspase-3,and Caspase-12 in the liver tissues of the model group was markedly up-regulated,and the expression of the anti-apoptotic protein Bcl-2 was markedly down-regulated;Treatment with HBOA significantly reversed the abnormal expression of these proteins.These results indicated that HBOA had a strong inhibitory effect on HFD-induced hepatocyte apoptosis in NAFLD rats.5.HBOA decreased hepatic lipid deposition in NAFLD ratsThe results of serum lipid levels showed that compared with the normal control group,the contents of FFA,TG,TC and LDL-C in the serum of the model group were significantly increased,and the content of HDL-C was significantly reduced;Compared with the model group,the HBOA treatment group had significantly reversed the disorder of serum lipid levels.The results of oil red O staining demonstrated that there were almost no red lipid droplets in the hepatocytes structural sequence of the normal control group,while the hepatocytes of the model group deposited a large number of red lipid droplets with different shapes.After HBOA treatment,it could significantly reduce the deposition of red lipid droplets in hepatocytes.These results indicated that HBOA could ameliorate HFD-caused disorder serum lipid levels and effectively reduce hepatic lipid accumulation in NAFLD rats.6.HBOA regulated the expression of hepatic lipid production-related genes and proteins in NAFLD ratsThe results of RT-PCR experiments demonstrated that compared with the normal control group,the m RNA expressions of SREBP-1c,FASN,and LXRαwere notably up-regulated and the m RNA expression of AMPK was notably down-regulated in the liver tissues of the model group;After treatment with HBOA,the abnormal expression of lipid synthesis genes in liver tissues was significantly reversed.Moreover,the Western blot results also confirmed that the expression levels of SREBP-1c,ACC1,FASN,and LXRα proteins were significantly up-regulated,and the phosphorylation level of AMPK protein was significantly down-regulated in the liver tissues of the model group;After treatment with HBOA,the abnormal expression of these lipid synthesis proteins could be significantly reversed.This evidence confirmed that HBOA could reduce the hepatic lipid deposition of NAFLD rats by regulating the expression of lipid production-related genes and proteins.7.HBOA inhibited the activation of ERS signaling pathway in NAFLD ratsThe results of RT-PCR and Western blot experiments showed that compared with the normal control group,the expression levels of the GRP78 gene and protein in the liver tissues of the model group were markedly up-regulated.After HBOA treatment,the abnormal expression of GRP78 could be markedly downregulated.Further experiments also demonstrated that long-term HFD can significantly up-regulate the phosphorylation levels of PERK,EIF2α,IRE1α,and JNK proteins in rat liver tissues,as well as the expression levels of ATF6,CHOP,ATF4,and XBP-1 proteins were significantly up-regulated.After HBOA intervention,the abnormal expression of these proteins can be significantly inhibited.Moreover,compared with the normal control group,the m RNA expression levels of PERK,EIF2α,CHOP,IRE1α,and ATF6 in the liver tissues of the model group were also significantly up-regulated,but after treatment with HBOA,the abnormal expression of these genes was significantly inhibited.Immunohistochemistry demonstrated that GRP78,PERK,EIF2α,CHOP and ATF6 proteins were only slightly expressed in liver tissue sections of the normal control group.Under the influence of long-term HFD,the expression of GRP78,PERK,e IF2α,CHOP and ATF6 was significantly increased,while the abnormal expression of these proteins was significantly reduced in the HBOA treatment group.These experimental results indicated that HBOA could alleviate HFDinduced NAFLD rats liver damage by inhibiting the activation of major proteins in the ERS signaling pathway.Conclusion:The results of this study demonstrate that HBOA can effectively relieve HFD-induced hepatocyte damage and hepatic steatosis in NAFLD rats,which may be attributed to the fact that HBOA can inhibit oxidative stress,enhancing antioxidant defense capabilities,alleviating inflammatory response,and decreasing the formation of lipid droplets.In addition,the investigation also demonstrates that HBOA can significantly ameliorate HFD-induced hepatic lipid metabolism disorders and hepatocyte apoptosis in rats by inhibiting the ERS signaling pathway and regulating the expression of lipid synthesis-related genes and proteins. |
