The Effect And Mechanism Of Raffinose On Improving Lipotoxic Liver Injury In Mice

Non-alcoholic fatty liver disease(NAFLD)is a nutritional metabolic syndrome of the liver caused by an imbalance in the metabolism of free fatty acids,and systematic research into the pathogenesis of the condition and the medications used to prevent and treat it is still lacking.Disorders of lipid metabolism are thought to be the initiating and pivotal factors in the development of NAFLD,but subsequent inflammation,pyroptosis,fibrosis and oxidative stress are also thought to play an important role in the development of the disease.Recent studies have shown that naturally occurring compounds can play an important role in the regulation of disorders of hepatic lipid metabolism through activation of the Nrf2 signaling pathway.Raffinose are naturally edible oligosaccharides that can be extracted from plants such as cottonseed and sugar beet and have biological functions such as being anti-inflammatory,antioxidant and regulating the metabolism of lipids.However,the potential role and molecular mechanism of cottonseed sugar in improving NAFLD is not yet clear.The Methionine and Choline Deficient Diet(MCD)results in impaired synthesis of phosphatidylcholine and consequently transport of triglycerides,ultimately resulting in impaired lipid synthesis and inducing NAFLD.At the same time,an in vitro model of lipotoxicity was constructed by treating AML12 cells with oleic acid(OA)and studied by liver histopathology,Oleic O stain,H&E stain,Sirius red stain,western blot(WB),RT-q PCR,and other methods.In order to elucidate the enhancing effect and mechanism of Raffinose on lipotoxicity,and provide potential drugs for prevention and control of lipotoxic liver injury in clinical practice.In vivo results demonstrated that raffinose could ameliorate MCD-induced histopathological liver injury,promoting expression of PPARα and inhibiting SREBP-1c expression,and regulating fatty acid metabolism.The results of WB showed that raffinose reduced the expression and secretion of inflammatory factors such as HMGB1,IL-6,and COX2 through down regulation of the TLR4-My D88-NF-κB signaling pathway.We also found that the cottonseed glycan inhibited NLRP3 inflammasome vesicle synthesis and reduced the shear inhibitory process of N-GSDMD.Staining with Sirius red revealed decreased deposition of collagen fibrils following raffinose treatment.α-SMA and TGF-β1,which are proteins related to fibrosis,changed in accordance with the staining results.The levels of Oxidative stress revealed that raffinose activated the Nrf2/Keap1 signaling pathway and promoted the expression of downstream antioxidant proteins such as Cav1 protein.In order to further validate the serological assay,GSH and SOD levels were decreased and MDA was increased.We conclude that cottonseed glycans were able to inhibit various aspects of disorders of lipid metabolism,inflammation,and oxidative stress,and ameliorate hepatic lipotoxic injury.In OA-induced AML12 cells,it was possible to find that cottonseed sugar attenuated abnormal lipid metabolism by regulating the expression of SREBP-1c,FAS,and PPARα.Oil Red O staining corresponded well with protein expression results.In partial agreement with in vivo experiments,we observed an effect of cottonseed sugar on the TLR4-My D88-NF-κB signaling pathway.Analysis of proteins associated with scorch death revealed that NLRP3,GSDMD,and Caspase-1 were significantly reduced by the cotton-sugar intervention.Furthermore,cottonseed sugar was capable of inducing activation of the Nrf2/Keap1 signaling pathway and promoting Nrf2 expression.Based on the above findings,it appears that cottonseed sugar can effectively ameliorate lipotoxic injury to hepatocytes and its function may be related to activation of the Nrf2/Keap1 signaling pathway.Subsequent experiments were carried out using Nrf2 knockout mice to investigate the relationship between Nrf2 and the cottonseed glycan.Observation of liver tissue,H&E staining,and Oil Red O staining showed that hepatic steatosis and lipid accumulation were increased following Nrf2 knockdown,and that the effect of treatment with cottonseed sugars was reduced.These findings were consistent with WB experiments.Using serological assays,we found that TG and TC clearance by Raffinose was impaired when Nrf2 was knocked-down.Further testing of the TLR4-My D88-NF-κB signaling pathway revealed that the inhibitory effect of the cottonseed glycan on the downstream IL-6 and TNF-α was diminished when Nrf2 was knocked down.NLRP3 and N-GSDMD proteins related to focal death showed trends similar to ASC.Sirius red staining also revealed that when deposition of collagen fibrils increased following Nrf2 knockdown,the ameliorative effect of cottonseed sugar was reduced.Protein expression related to fibrosis was consistent with the staining results.In conclusion,WB experiments and serological assays revealed that HO-1 and NQO1 expression were inhibited when Nrf2 knockdown was performed,decreased levels of GSH and SOD in serum,and a decreased regulatory effect of the raffinose.Thus,we hypothesize that cottonseed sugar’s amelioration of hepatic lipotoxic injury may be linked to Nrf2.In conclusion,this experiment elucidated the mechanism of action of cottonseed sugar in the amelioration of NAFLD through the construction of an in vivo and ex vivo model of lipid metabolism disorder.Our results demonstrated that Raffinose improved the development of NAFLD by targeting the Nrf2 signaling pathway and thus inhibiting disorders of lipid metabolism,inflammation,burning,and oxidative stress.