| Objective To investigate the effects of total saponins from Panax japonicus on adipose tissue inflammation induced by high-fat diet and palmitic acid(PA)-induced mature adipocyte inflammation,and further study the underlying molecular mechanisms.This study will provide a new idea for the treatment of metabolic diseases caused by high-fat diets.Methods 1.In vivo experiment: Fifty Babl/c male mice were randomly divided into normal diet group,high-fat model group,low,medium and high doses group of total saponins from Panax japonicus.10 mice per group,each mouse weighs 18-20 g.Low,medium and high doses of total saponins from Panax japonicus were given by gastric gavage of total saponins from Panax japonicus 30 mg/kg,90 mg/kg,150 mg/kg,respectively.The normal diet group and the high-fat model group were given 0.5% sodium carboxymethyl cellulose and withdrawal 1 day a week,lasting for 4 months.During the administration,the mice in the normal diet group were given normal diet,and the high-fat model group and the low,medium,and high dose groups of the total saponins from Panax japonicus were given high-fat diet,each group of mice was given a free diet.During feeding period:(1)Body weight of mice in each group were recorded everyday.And we underwent oral glucose-tolerance test once a month.At the end of treatment,the mice were euthanized,and serum sample were collected.Observed the following indicators:(2)The visceral fat were obtained to calculate visceral fat weight and visceral fat index.(3)The levels of glucose,triglyceride,cholesterol,high density lipoprotein-cholesterol,low density lipoprotein-cholesterol in serum were measured by the commercial kits according to the protocols.(4)Pathological changes of epididymal adipose tissue were stained with hematoxylin and eosin(HE).(5)The relative mRNA levels of TNF-?,IL-1?,iNOS and MCP-1 in adipose tissue were detected by real-time quantitative PCR.(6)The relative protein levels of TNF-?,IL-1? and iNOS in adipose tissue were detected by Western blot.(7)The expression of pNF-?B/NF-?B,pI?B/I?B signaling pathway proteins were detected by Western blot.(8)The relative mRNA levels of M1 macrophage markers F4/80 and CD11 b in adipose tissue were detected by real-time quantitative PCR.(9)The proportion of macrophages in epididymal adipose tissue of each group was detected by flow cytometry.2.In vitro experiments:(1)The effects of total saponins from Panax japonicus on the proliferation of 3T3-L1 preadipocytes was detected by Ethylthiazolyldiphenyl-tetrazolium bromide(MTT).(2)3T3-L1 preadipocytes were induced into mature adipocytes with traditional "cocktail" method.Oil red O staining was used to observed whether 3T3-L1 preadipocytes were differentiated into mature adipocytes successfully.(3)The mature adipocytes were treated with PA and different concentrations of total saponins from Panax japonicas.(4)The relative protein levels of TNF-?,IL-1? and iNOS were detected by Western blot.(5)The expression of pNF-?B/NF-?B,pI?B/I?B signaling pathway proteins were detected by Western blot.Results 1.In vivo experiments:(1)The mouse obesity model was established successfully.During the feeding period,the weight gain of the mice in each group was significantly different.Compared with normal diet group,the mice in high-fat model group showed a significant increase in body weight from 9 weeks of age(p < 0.01).Compared with high-fat model group,the weight gain rate of the mice in each drug group was significantly decreased in different feeding period(p < 0.05 or p < 0.01).(2)Compared with normal diet group,the visceral fat index of high-fat model group was significantly increased(p < 0.05).However drug groups decreased the visceral fat index.(3)After administration of glucose for 0.5 h,the blood glucose level of high-fat model group was significantly higher than that of normal diet group(p < 0.01).Compared with high-fat model group,the blood glucose level was decreased after total saponins from Panax japonicus treatment(p < 0.05).During the administration of glucose for 0.5 h to 2.5 h,the blood glucose levels of high-fat model group were significantly higher than that of normal diet group(p < 0.01).After treatment with total saponins from Panax japonicus,the blood glucose levels of the drug groups were lower than that of high-fat model group(p < 0.05 or p < 0.01).(4)Compared with normal diet group,the content of glucose,triglyceride,cholesterol,and low density lipoprotein-cholesterol in serum of high-fat model group were significantly increased(p < 0.05),and the high density lipoprotein-cholesterol content was significantly decreased(p < 0.05).Conversely,the content of glucose,triglyceride,cholesterol,and low density lipoprotein-cholesterol in serum was decreased,and the high density lipoprotein-cholesterol content was increased after total saponins from Panax japonicus treatment.(5)The results of HE staining showed that fat cells in normal diet group were closely arranged,generally round and uniform in size.Compared with normal diet group,the fat cells of high-fat model group were disordered in structure,uneven in size,and scattered,the crown-like structure formed by macrophage infiltration was significantly increased(p < 0.01).The total saponins from Panax japonicus significantly improved the cell morphology,reduced macrophage infiltration caused by high-fat diet(p < 0.05).(6)The results of real-time quantitative PCR and Western blot showed that the expressions of TNF-??IL-1??iNOS and MCP-1 in adipose tissue of high-fat model group were significantly higher than that of normal diet group.The expression levels of TNF-??IL-1??iNOS and MCP-1 were significantly decreased after the treatment with total saponins from Panax japonicus.(7)Western blot results showed that the protein expression levels of pNF-?B and pI?B in adipose tissue of the high-fat model group were significantly higher than that of normal diet group(p < 0.01),and after total saponins from Panax japonicus treatment,the expression of pNF-?B and pI?B were significantly decreased(p < 0.05).The protein expression levels of NF-?B,I?B and IKK showed no significant difference in each group.(8)The results of real-time quantitative PCR showed that the mRNA expression levels of F4/80 and CD11 b in the adipose tissue of the high-fat model group were significantly higher than that of normal diet group(p < 0.01).The expression levels of F4/80 and CD11 b were significantly decreased after the treatment with total saponins from Panax japonicus(p < 0.01).(9)The results of flow cytometry showed that the proportion of mature macrophages in the visceral adipose tissue of high-fat model group was significantly higher than that of normal diet group,and the proportion of macrophages in the visceral adipose tissue of each drug group were lower than that of high-fat model group.2.In vitro experiments:(1)successfully induced 3T3-L1 preadipocytes into mature adipocytes,and lipid droplets in mature adipocytes were stained red by oil red O;(2)The results of MTT showed that the cell proliferation was significantly inhibited when the concentration of total saponins from Panax japonicus was higher than 50 ?g/ml(p < 0.05 or p < 0.01).When the concentration was less than 50 ?g/ml,the cell proliferation of 3T3-L1 preadipocytes was not affected.(3)Western blot results showed that compared with the normal group,the protein expression level of TNF-?,IL-1? and iNOS were significantly increased in the PA-treated model group(p < 0.01),and after total saponins from Panax japonicus treatment,the expression of TNF-?,IL-1? and iNOS were significantly decreased(p < 0.05).(4)Western Blot results showed that the protein expression levels of pNF-?B and pI?B in the PA-treated model group were significantly higher than that of normal group(p < 0.01),but compared with the PA-treated group,the protein expression levels of pNF-?B and pI?B in total saponins from Panax japonicus treatment group were significantly decreased(p < 0.01).Among them,the protein expression levels of NF-?B,I?B and IKK in each group were no significant difference.Conclusion Total saponins from Panax japonicus can significantly improve chronic inflammation of adipose tissue induced by high-fat diet and PA-induced mature adipocyte inflammation.The mechanism may be related to reducing macrophage infiltration and inducing the transformation of pro-inflammatory macrophages to anti-inflammatory macrophages.The inhibition of NF-?? inflammatory pathway is also involved in the mechanism. |
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