| Section 1 The effect of berberine on inhibiting adipose tissue fibrosis induced by high-fat diet in obese miceObjectiveThe present study aimed to investigate the effect and the underlying mechanism of low,medium and high doses of berberine on adipose tissue fibrosis in high-fat diet induced obese mice.MethodsMale C57BL/6 mice were fed with 60% high-fat diet for 16 weeks to establish obese mice model.The mice were randomly divided into high-fat-diet group and three different doses of berberine(100 mg/kg,200 mg/kg and 300 mg/kg)treatment groups.The normal chow diet group was set as normal control.The differences of body weight,fasting blood glucose,GTT and ITT were measured in the groups.HE staining was used to observe the morphological changes in white adipose tissue of epididymis in mice;Masson’s Trichrome staining and Sirius Red staining were used to detect collagen deposition in epididymal white adipose tissue;RT-q PCR was used to detect the gene transcription levels of Collegen I,Fibronection and α-SMA in epididymal white adipose tissue;Western blot was used to detect Collegen I,α-SMA and PDGFR-α in white adipose tissue of epididymis.The expression levels of fibronection and α-SMA in epididymal white adipose tissue were detected by immunohistochemistry.Western blot was used to detect the expression of HIF-1α in epididymal white adipose tissue of epididymis,and RT-q PCR was used to detect LOX gene transcription.Western blot was used to detect cleaved caspase 3 protein expression in epididymal white adipose tissue.The protein expression of cleaved caspase 3 in epididymal white adipose tissue was detected by immunohistochemistry.ResultsThe results of body weight,GTT and ITT showed that berberine can reduce body weight and regulate glucose metabolism in a dose dependent manner.Compared with the normal diet group,the cell size of epididymal white adipose tissue in HFD mice increased significantly.BBR treatment significantly reduced the cell size of epididymal adipose tissue.Masson’s Trichrome staining and Sirius Red staining showed that white adipocyte clusters in epididymal adipose tissue of HFD mice were segmented by bundle and rope collagen,and collagen deposition was increased.After BBR treatment,collagen deposition decreased significantly.Compared with normal diet group,the gene transcription of Collegen I,Fibronection and α-SMA in epididymal adipose tissue of HFD group mice was enhanced.However,BBR reduced the m RNA expression of these genes.Compared with the normal diet group,the protein expression of Collegen I,α-SMA,PDGFR-α and Fibronection in HFD group was increased,and BBR treatment could inhibit the expression of these proteins.Compared with the normal diet group,the protein expression of HIF-1 α in epididymal white adipose tissue of HFD mice was increased,and the expression of HIF-1α in e WAT of mice was inhibited by BBR treatment.Compared with mice in the normal diet group,as an important target gene of HIF-1α,the m RNA expression of lysyl oxidase(LOX)gene catalyzing collagen modification and cross-linking in epididymal adipose tissue of mice in the HFD group was also enhanced,while BBR decreased the expression of LOX.In addition,BBR therapy attenuated cleaved caspase-3 protein expression in epididymal adipose tissue induced by HFD.ConclusionsBerberine can regulate the metabolic homeostasis,reduce collagen deposition and extracellular matrix composition secretion in epididymal white adipose tissue and alleviate adipose tissue fibrosis in obese mice induced by high-fat diet.The mechanism may be related to inhibiting the activation of HIF-1α in epididymal white adipose tissue.Section 2 The effect of Berberine on improving hyperlipidemia by inhibiting intestinal lipid absorptionObjectiveThe present study aimed to explore the effect and the underlying mechanism of berberine on regulating intestinal lipid absorption by using obese animal models induced by high fat diet and fatty acid absorption models induced by oleic acid in intestinal epithelial cells of Caco-2 and FHs 74 Int.MethodsMale C57BL/6 mice were fed with 60% high-fat diet for 16 weeks to establish obese mice model.The mice were randomly divided into high-fat diet group and different dose of berberine treatment groups.The normal chow diet group was set as normal control.The weight and food intake of mice were recorded during drug intervention.GTT,ITT and blood lipid profile were measured to evaluate the glycolipid metabolism of mice.HE staining and oil red O staining were used to observe the morphological changes of liver in mice.HE staining was used to observe the morphological changes of epididymal white adipose tissue,and immunohistochemical method was used to detect the expression of F4/80 in epididymal white adipose tissue of mice.HE staining was used to observe the morphological changes of the proximal small intestine.Oil red O staining was used to detect the lipid deposition in the proximal small intestine after 30 minutes of olive oil administration.Western blot analysis of CD36,FATP4,m TOR,NPC1LL1 and Numb.For combined treatment of oleic acid with BBR,Caco-2 cells were pretreated with 10,20 and 30 μM berberine for 30 minutes and then treated with 100 m M oleic acid for 24 hours.Then the absorption of fatty acids in Caco-2 cells was detected by BODIPY staining.The expression levels of CD36 and FATP4 were detected by Western blot.For combined treatment of oleic acid with BBR or rapamycin(m TOR inhibitor),FHs 74 Int cells were pretreated with 10μM,20μM,30μM berberine or 50 nM rapamycin for 30 minutes and then treated with 100 m M oleic acid for 24 hours.Then the absorption of fatty acids in FHs 74 Int cells was detected with BODIPY staining.The protein expression levels of m TOR,CD36 and FATP4 were detected by Western blot.ResultsThe results of body weight and food intake showed that berberine can reduce the weight of obese mice induced by high fat diet without affecting their food intake.The results of GTT and ITT showed that berberine can improve glucose tolerance and insulin resistance in obese mice.Compared with the normal diet group,adipocyte size,inflammatory infiltration and liver fat deposition were increased in the high fat diet group.Berberine could significantly improve the metabolic disorders in these peripheral tissues.There was no significant difference in the length and histomorphology of the proximal small intestine between groups.Compared with the normal diet group,the lipid deposition in the proximal small intestine increased in the high fat diet group,and BBR could significantly inhibit the lipid deposition in the proximal small intestine.BBR increased the excretion of fecal triglycerides in mice fed with high-fat diet.Compared with normal diet group,the protein expression of m TOR,CD36,FATP4,NPC1L1 and Numb in proximal small intestine of mice fed with high fat diet was increased significantly,while the expression of above proteins in berberine treatment group was decreased significantly.Lipid deposition was obvious in Caco-2 cells and FHs 74 Int cells treated with oleic acid.Berberine could inhibit lipid deposition in these cells.Compared with the control group,the fatty acid transporters of CD36 and FATP4 in Caco-2 cells and FHs 74 Int cells of oleic acid intervention group were inhibited by berberine,and rapamycin inhibited the expression of CD36 and FATP4 in FHs 74 Int cells.ConclusionsBerberine can effectively reduce intestinal lipid absorption and increase fecal triglyceride excretion in high-fat-induced animal models,thus reducing blood lipid levels,and down-regulating the expression of cholesterol transport-related proteins NPC1L1 and Numb,as well as down-regulating the expression of fatty acid transport-related proteins CD36,FATP4 and m TOR.Cell experiments further confirmed that berberine inhibited fatty acid uptake and decreased the expression of fatty acid transporters CD36 and FATP4 in intestinal epithelial cells,and that berberine may regulate the expression of m TOR. |
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