| Fatty liver is a metabolic disease with high incidence in high-yield animals of modern animal husbandry,which reduces production and reproduction performance,damages animal welfare and brings huge economic losses.However,there is no effective specific regulatory substance to prevent fatty liver.Salidroside(SAL)is a plant extract with high efficiency and low toxicity,and has a variety of pharmacological activities.The previous study of our research group found that the incidence of fatty liver disease in perinatal dairy cows was as high as 58.6%.As a large animal,in the study of fatty liver,it is difficult to sample dairy cattle livers in vivo,which affects animal production,and the cost of slaughter sampling is high.Model animal mice have been widely used to study the mechanism of liver physiology or pathology.Therefore,this study investigated the regulation of salidroside on lipid deposition and inflammation in fatty liver model through cell test and mouse test.Through transcriptome technology,the mechanism of salidroside was explored and analyzed.Gene silencing technique and specific inhibitors were used to verify the screened pathway in vitro and in vivo.This study aims to provide theoretical basis and new ideas for the prevention and control of fatty liver in dairy cows during animal production.Part one Regulation of salidroside on fat deposition and inflammatory responseCell test: Palmitic/oleic acid(PO)stimulated primary mouse hepatocytes for 12 h to simulate lipid deposition and inflammatory response induced by lipid metabolites during fatty liver progression.Primary hepatocytes were divided into four groups:control group,model group(only PO-treatment),SAL-L group(PO+SAL 50 μmol/L)and Sal-H group(PO+SAL 100 μmol/L).Nile red staining was performed in each group,and the transcriptome sequencing analysis were performed in model group and SAL-H group.The main results are as follows:(1)Nile red staining showed a obvious increase in orange lipid droplets in the model group compared with the control group.Compared with the model group,the accumulation of lipid droplets in the SAL-L and SAL-H groups was reduced in a dose-dependent manner.(2)Transcriptome results showed that the expression of cellular lipid metabolism and inflammation-related signal transduction and molecules were down-regulated in the SAL-H group compared with the model group.Mice test: forty SPF grade,8～10-week-old,22g～25 g C57BL6/J male mice were divided into four groups: NC-Vehicle group(control group),HFHC-Vehicle group(model group),HFHC-Sal-L group(HFHC-diet+SAL 100 mg/kg/d)and HFHC-Sal-H group(HFHC-diet+SAL 200 mg/kg/d).Mice were fed a high fat/high cholesterol(HFHC)diet for 16 weeks to establish steatohepatitis model,and the control group was fed a normal diet.From the 9th week,mice in the HFHC-Sal-L and HFHC-Sal-H groups were fed HFHC diet and orally gavaged with corresponding doses of salidroside every day for 8 weeks.The results are as follows:(1)Compared with the NC-Vehicle group,fasting body weight(BW),white fat weight(WW),and white fat weight/body weight(BW/WW)of HFHC-Vehicle mice were significantly increased(P<0.01).Compared with HFHC-vehicle group,BW,WW and WW/BW of mice in HFHC-Sal-L and HFHC-Sal-H groups were significantly decreased(P<0.01).(2)H&E and oil red O staining showed that salidroside reduced cytoplasmic fat vacuole,balloon degeneration and lipid droplet accumulation in liver induced by HFHC diet in a dose-dependent manner.Compared with NC-Vehicle group,liver weight,liver-body-ratio,liver triglycerides(TG)and total cholesterol(TC)of mice in HFHC-Vehicle group were significantly increased(P<0.01).Compared with HFHC-Vehicle group,liver weight,liver-body-ratio,TG and TC were significantly decreased in HFHC-SAL-L and HFHC-SAL-H groups(P<0.01).(3)Compared with HFHC-Vehicle group,m RNA(CD36,FABP1,FASN,PPARγ,SCD1 and SREBP-1)and protein(FASN and PPARγ)expression of lipid-metabolism related genes were significantly decreased in HFHC-SAL-L and HFHC-SAL-H groups(P<0.01).(4)In immunohistochemistry sections,salidroside reduced the infiltration of Cd11b-positive cells in liver tissue induced by the HFHC diet.Compared with HFHC-Vehicle group,the m RNA expression levels of pro-inflammatory cytokines(IL-1β,IL-6 and TNF-α)and chemokines(CCL2 and CXCL10)in HFHC-SAL-L and HFHC-SAL-H groups were significantly decreased(P<0.01),IKKβ and P65 protein phosphorylation levels were significantly decreased(P<0.01),and IKBα protein expression level was significantly increased(P<0.01).(5)Transcriptome results showed that the expression of lipid metabolism and inflammation-related signal transduction and molecules were down-regulated in liver of HFHC-Sal-H group compared with HFHC-Vehicle group.Part two Mechanism of salidroside regulating lipid deposition and inflammationMechanism exploration: to clarify the specific mechanism of salidroside alleviating fat deposition and inflammation,transcriptome data of PO-stimulated primary hepatocytes and liver tissues of HFHC-diet mice with salidroside treatment were analyzed,and protein expression of the screening pathway was detected by Western Blot.The results are as follows:(1)KEGG enrichment analysis showed that the regulation of salidroside in cells and mice involved multiple signaling pathways,and AMPK signaling pathway was the most significantly changed in the common pathway.(2)Western Blot results demonstrated that salidroside significantly enhanced phosphorylation of AMPKα in PO-stimulated primary hepatocytes and liver of HFHC-diet mice(P<0.01),promoting the activation of AMPK signaling pathway.Cell verification: in order to verify that salidroside alleviates lipid deposition and inflammation of in vitro model of fatty liver through AMPK signaling,AMPK gene silencing and salidroside treatment were performed in PO-stimulated primary hepatocytes,which were divided into: Adsh RNA-DMSO group(PO stimulation),Adsh RNA-Sal group(PO+SAL),Adsh AMPK-DMSO group(PO+AMPK kockdown)and Adsh AMPK-Sal group(PO+AMPK kockdown + SAL).The results were as follows:(1)Nile red staining showed that PO-stimulated orange lipid droplets decreased significantly in Adsh RNA-Sal group compared to Adsh RNA-DMSO group,while the Adsh AMPK-Sal group was comparable to those in the Adsh AMPK-DMSO group.(2)Compared with Adsh RNA-DMSO group,hepatocyte TG content,m RNA expression levels of fatty acid synthesis(SCD1,ACC and FASN)and inflammation related genes(IL-1β,IL-6 and TNF-α)were significantly decreased in Adsh RNA-Sal group(P<0.01),while the above indexes showed no significant difference between Adsh AMPK-Sal group and Adsh ANPK-DMSO group(P>0.05).Animal verification: to verify that salidroside alleviates lipid deposition and inflammation in mouse model of fatty liver through AMPK,HFHC-diet mice were fed compound C(CC)to inhibit AMPK expression,and salidroside was administered orally.Forty SPF grade,forty 8～10-week-old,22g～25g C57BL6/J male mice were divided into 4 groups: PBS-Vehicle group(only HFHC-diet),PBS-Sal-H(HFHC-diet+SAL 200mg/kg/d),CC-Sal-H(HFHC-diet+CC+SAL 200mg/kg/d),CC-Vehicle(HFHC-diet+CC).The test results are as follows:(1)H&E and oil red O staining and immunohistochemistry results showed that the liver cytoplasmic fat vacuole,balloon degeneration and lipid drop accumulation as well as the infiltration of Cd11 b positive cells were significantly reduced in the PBS-Sal-H group compared with the PBS-Vehicle group,and the staining results between CC-Sal-H and CC-Vehicle groups were similar.(2)Compared with PBS-Vehicle group,liver weight,liver-body-ratio,liver TG and TC,m RNA levels of fatty acid intake(CD36,FASN,PPARγ and SREBP-1)and inflammation(IL-1β,IL-6 and TNF-α)related genes in liver were significantly decreased in PBS-Sal-H group(P<0.01),while the above indexes showed no significant difference between CC-Vehicle and CC-Sal-H groups(P>0.05).In conclusion,salidroside alleviates lipid deposition and inflammatory response in vivo and in vitro models of fatty liver by enhancing phosphorylation of AMPKαand promoting activation of AMPK signaling pathway. |
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