| ObjectiveMicroglia play a crucial role in neuroinflammation.The activation of hypothalamic microglia and inflammatory reactions induced by chronic calorie excess are associated with the occurrence and development of obesity.As a member of the nicotinamide adenine dinucleotide(NAD+)dependent enzyme Sirtuin family,Sirtuin 6(Sirt6)consists of three forms of enzyme activity,namely deacetylase,adenosine diphosphate transferase,and degrease acyltransferase.The demand of Sirt6 for NAD+during deacetylation closely links Sirt6 with metabolism and nutritional status.As suggested in existing studies,Sirt6 is an important regulatory factor involved in energy metabolism in many anorexia neurons in the peripheral tissue and hypothalamus.However,the effect of Sirt6 on hypothalamic inflammation related to obesity induced by diet has not been reported.Considering the important role of microglia in hypothalamic inflammation related to diet-induced obesity,the effect of Sirt6 in microglia on diet-induced obesity was explored by constructing mice with the conditioned knockout of microglial Sirt6.According to traditional Chinese medicine,phlegm stagnation is the key pathogenesis of obesity.Some conditions such as stagnation and heat may be caused over time,which may induce more complex lesions.Danggui-Shaoyao-San(DSS)is derived from Jinkui Yaolue,and it is composed of Angelica sinensis,Paeonia lactiflora,Ligusticum wallichii,Poria cocos,Atractylodes macrocephala Koidz and Alismatis rhizoma.This prescription is effective in regulating the liver and spleen simultaneously,and it has the effects of promoting blood circulation,removing blood stasis,promoting diuresis,eliminating turbidity,and resolving phlegm.The results of previous studies on DSS demonstrated that DSS could mitigate lipid metabolic disorders.DSS water extract down-regulated the expression of TC,TG,and Low Density Lipoprotein Cholesterol(LDL-C)in APP/PS1 double transgenic AD model mice.Besides,DSS can regulate the inflammatory response of the central nervous system.In ovariectomized mice,DSS was found to play a neuroprotective role by inhibiting hippocampal inflammation.In view of the fact that obesity is accompanied by glucolipid metabolic disorders in peripheral tissues and neuroinflammatory reactions in the central nervous system,an obesity model induced by a high-fat diet(HFD)was established in this study.Moreover,the therapeutic effect and mechanism of DSS on obesity were preliminarily investigated.MethodsExperiment I:Sirt6 in hypothalamic microglia responded to changes in the nutritional environment:(1)Mice were randomly divided into three groups,namely the normal diet group,the HFD-4-week group,and the HFD-8-week group,with 6 mice in each group.Mice in the normal diet group received a normal diet,while those in the HFD group received a high-fat diet.At the end of the experiment,the heart of mice was perfused first,and the whole brain was collected.Immunofluorescence was performed to detect the expression of Sirt6 in mice hypothalamic microglia.(2)In another experiment,mice were randomly divided into two groups,namely the normal diet group and the HFD-24-week group,with 6 mice in each group.Mice in the normal diet group received a normal diet,while those in the HFD group received a high-fat diet.At the end of the experiment,the primary microglia of mice was collected.Subsequently,the mRNA expression level of Sirt6 in primary microglia was detected by qPCR.(3)Primary microglia from newborn mice within 24 h were isolated and cultured.Then,the cells were treated with Oleic Acid(OA)&Palmitic Acid(PA)for 24 h.Next,the protein expression level of Sirt6 in primary microglia was detected.(4)BV2 cells were treated with OA&PA for 12 h or 24 h,and the mRNA and protein expression level of BV2 cells was detected.Experiment Ⅱ:Sirt6 improved the inflammatory response of BV2 cells induced by OA&PA treatment:(1)In BV2 cells,Sirt6 was overexpressed by adenovirus transfection.Next,these cells were treated with OA&PA for 24 h.After that,the immunofluorescence assay was employed to detect the expression of CD68 and TNF-α in BV2 cells.(2)In BV2 cells,Sirt6 was overexpressed by adenovirus transfection.Next,these cells were treated with OA&PA for 24 h.Then,the mRNA expression of Tnf-α,Interleukin-6(Il-6),and Interleukin-1β(Il-1β)in BV2 cells was detected by qPCR.(3)Sirt6 was overexpressed by adenovirus transfection in BV2 cells.Subsequently,these cells were treated with OA&PA for 24 h.Then,the Enzyme-Linked ImmunoSorbent Assay(ELISA)was used to detect the expression of TNF-α,IL-6,and IL-1β in the cell culture medium.Experiment III:Sirt6 in hypothalamic microglia was involved in the regulation of HFDinduced obesity:Firstly,SIRT6flox/folx mice(SIRT6 fl/fl mice)were hybridized with Cx3cr1Cre mice to obtain the conditioned knockout of microglial Sirt6 mice(SIRT6Mic-/-mice).Subsequently,SIRT6fl/fl mice(6-8 weeks old)and SIRT6Mic-/-mice(6-8 weeks old)received HFD for 12 weeks.In contrast,SIRT6fl/fl mice and SIRT6Mic-/-mice of the same age were selected as controls to receive a normal diet.The Inguinal White Adipose Tissue(iWAT)and Epididymal White Adipose Tissue(eWAT)of these mice were reconstructed by CT scanning.The morphology of the liver,iWAT,eWAT,and brown adipose tissue(BAT)were observed under hematoxylin-eosin staining(HE staining).The lipid accumulation in the liver of mice was observed under oil red O staining.In addition,immunofluorescence staining was used to detect the expression of ibal in the hypothalamus of mice.Moreover,the mRNA expression of inflammatory cytokines(Tnf-α,Il-6,and Il-1β)in the hypothalamus of mice was detected by qPCR.Experiment Ⅳ:DSS exerted effects on peripheral glucolipid metabolism and hypothalamic inflammation induced by HFD in obese mice:Mice were randomly divided into 5 groups,namely the normal diet group,the HFD group,the pioglitazone hydrochloride group,the low-dose-DSS group,and the high-dose-DSS group,with 16 mice in each group.Mice in the normal diet group received a normal diet,while those in the HFD group received a high-fat diet.In the experiment,model construction and administration were performed at the same time.The dosage in the low-dose-DSS group and the high-dose-DSS group was 3.2 g·kg-1·d-1 and 6.4 g·kg-1·d-1,respectively.The dosage in the pioglitazone hydrochloride group was 10 mg·kg-1·d-1.Mice in the control group and the model group were administered with 10 ml·kg-1·d-1 normal saline.All mice received intragastric administration once a day for 12 weeks.The body mass of mice was recorded every week.After HFD,all mice underwent the glucose tolerance test(GTT),insulin tolerance test(ITT),and pyruvate tolerance test(PTT).The iWAT and eWAT of mice were reconstructed by CT scanning.The morphology of the liver,iWAT,eWAT,and BAT was observed under HE staining.The lipid accumulation in the liver of mice was observed under oil red O staining.In addition,immunofluorescence staining was used to detect the expression of ibal and Sirt6 in the hypothalamus of mice.Moreover,the expression of inflammatory cytokines(TNF-α,IL-6,and IL-1β)in the hypothalamus of mice was detected by the Elisa assay.ResultsExperiment Ⅰ:Sirt6 in hypothalamic microglia responded to changes in the nutritional environment.(1)Compared with the normal diet group,8 weeks of HFD exposure induced slight down-regulated expression of Sirt6 in hypothalamic microglia.(2)Compared with the normal diet group,24 weeks of HFD exposure induced down-regulated mRNA expression of Sirt6 in hypothalamic microglia.(3)OA&PA treatment down-regulated the protein expression level of Sirt6 in primary microglia.(4)OA&PA treatment down-regulated the mRNA and protein expression of Sirt6 in BV2 cells.Experiment Ⅱ:Overexpressed Sirt6 alleviated the inflammatory response of BV2 cells induced by OA&PA treatment.(1)In BV2 cells,the expression of CD68 and TNF-α was upregulated by OA&PA treatment,and that of CD68 and TNF-α was down-regulated by the overexpression of Sirt6.(2)In BV2 cells,OA&PA treatment significantly up-regulated the mRNA expression of inflammatory cytokines(Tnf-α,Il-6,and Il-1β),and overexpressed Sirt6 significantly down-regulated the mRNA expression of Tnf-α,Il-6,andIl-1β.(3)In BV2 cells,OA&PA treatment significantly up-regulated the expression of inflammatory cytokines,and overexpressed Sirt6 significantly down-regulated the expression of inflammatory cytokines.Experiment Ⅲ:Sirt6 in hypothalamic microglia was involved in the regulation of obesity induced by HFD.(1)The knockout of Sirt6 in microglia aggravated obesity induced by HFD.In the normal diet group,mice with the knockout of Sirt6 in microglia had a slightly increased body weight,but the difference was negligible.Similarly,the weight of iWAT,eWAT,and BAT also increased slightly,but the difference was negligible.However,the HE staining results showed that the knockout of Sirt6 in microglia led to morphological hypertrophy of iWAT cells in mice.In the HFD group,the knockout of Sirt6 in microglia resulted in a significant increase of body weight and the adipose tissue in mice.Compared with SIRT6fl/fl mice after 12 weeks of HFD,SIRT6Mic-/-mice had a significantly increased body weight during the same period.Similarly,the adipose tissue of SIRTMic-/-mice significantly increased compared with SIRT6fl/fl mice.This finding was confirmed by MicroCT images of iWAT and eWAT,the weight of iWAT,eWAT,and BAT,as well as the HE staining images of iWAT,eWAT,and BAT.Additionally,the liver/body weight ratio of SIRT6Mic-/-mice was slightly higher than that of SIRT6fl/fl mice,but the difference was not statistically significant.HE staining of liver indicated that SIRT6Mic-/-mice showed aggravated balloon-like degeneration.Moreover,liver oil red O staining indicated that SIRT6Mic-/-mice showed more liver lipid accumulation and more severe liver steatosis after 12 weeks of HFD.(2)The knockout of Sirt6 in microglia aggravated HFD-induced hypothalamic inflammation.The immunofluorescence staining results of brain tissues showed that there was no significant difference in the number and size of hypothalamic microglia between SIRT6fl/fl mice and SIRT6Mic-/-mice in the normal diet group.Moreover,the qPCR results demonstrated that there was no significant difference in the mRNA expression of inflammatory cytokines(Tnf-α,Il-6,and Il-1β)between SIRT6fl/fl mice and SIRT6Mic-/-mice in the normal diet group.In mice in the HFD group,the immunofluorescence staining results revealed that knockout of Sirt6 in microglia aggravated HFD-induced hypothalamic microglial activation.Furthermore,the mRNA expression of inflammatory cytokines(Tnf-α,Il-6,and Il-1β)in the hypothalamus of SIRT6Mic-/-mice in the HFD group was up-regulated compared with SIRT6fl/fl mice in the same groups.Experiment Ⅳ:DSS alleviated the obesity and hypothalamic inflammation induced by HFD in mice.(1)DSS reduced the body mass and adipose tissue weight of obese mice induced by HFD and improved the blood lipid status of these mice.Compared with the control group,the body weight and adipose tissue weight of mice in the model group increased significantly.Pioglitazone hydrochloride had no significant influence on the body weight and adipose tissue weight of HFD mice.DSS significantly reduced the body weight and adipose tissue weight of mice,which was more significant in the high-dose-DSS group compared with the low-dose-DSS group.The HE staining results demonstrated that compared with the control group,morphological hypertrophy of iWAT and eWAT cells occurred in the model group.Besides,"whitening" was also observed in BAT.Pioglitazone hydrochloride had no significant influence on the morphology of iWAT,eWAT,and BAT in HFD mice.Both low and high doses of DSS significantly reduced the hypertrophy of iWAT and eWAT cells in mice and mitigated the "whitening" of BAT.Additionally,the HE staining results of the liver indicated that compared with the control group,mice in the model group presented aggravated balloon-like degeneration.Pioglitazone hydrochloride,low doses of DSS,and high doses of DSS can alleviate balloon-like degeneration in the liver of mice.Similarly,the oil red O staining results of the liver indicated that compared with the control group,mice in the model group presented more lipid accumulation and more severe steatosis in the liver of mice.Pioglitazone hydrochloride,low doses of DSS,and high doses of DSS can alleviate lipid accumulation and steatosis in the liver of mice.Among them,the optimal effect can be observed in the high-dose-DSS group,followed by the pioglitazone hydrochloride group.Compared with the control group,the expression of serum TG,TC,and LDL-C in the model group was significantly up-regulated,while that of HDL-C was significantly down-regulated.Pioglitazone hydrochloride,low doses of DSS,and high doses of DSS can down-regulate the expression of serum TG,TC,and LDL-C and up-regulate that of serum HDL-C in mice.Additionally,DSS alleviated obesity-related pathological injuries in mice.Compared with the control group,the AUC of GTT,ITT,and PTT in the model group significantly increased.Pioglitazone hydrochloride,low doses of DSS,and high doses of DSS can significantly reduce the AUC of GTT,ITT,and PTT in mice.These results indicated that pioglitazone hydrochloride,low doses of DSS,and high doses of DSS could alleviate glucose tolerance impairment,insulin resistance,and gluconeogenesis in the liver of mice.(2)DSS up-regulated the expression of Sirt6 in microglia in the ARC region of the hypothalamus and mitigated the inflammatory reaction in the hypothalamus of obese mice.The immunofluorescence staining results showed that microglia were activated in the ARC region of the hypothalamus in HFD mice.The administration of DSS reduced activated microglia in the ARC region.Besides,DSS also up-regulated the expression of Sirt6 in microglia.Compared with the control group,the expression of Sirt6 in microglia in the ARC region of the hypothalamus in HFD mice was slightly down-regulated.Pioglitazone hydrochloride had no significant influence on the expression of Sirt6 in the microglia of HFD mice.The expression of Sirt6 in microglia was up-regulated in the low-and high-dose-DSS groups.The finding that DSS alleviated hypothalamic inflammatory responses in HFD mice was further verified by the ELISA results.Compared with the control group,the expression of TNF-α,IL-6,and IL-1β in the hypothalamus of the model group was up-regulated.Pioglitazone hydrochloride had no significant influence on the expression of TNF-α,IL-6,and IL-1β in the hypothalamus of HFD mice.The expression ofTNF-α,IL-6,and IL-1 β in the hypothalamus of mice was down-regulated by both low and high doses of DSS.Conclusions(1)Sirt6 in hypothalamic microglia was involved in the regulation of obesity induced by HFD.The knockout of Sirt6 in microglia aggravated obesity induced by HFD in mice.Moreover,the knockout of Sirt6 in microglia aggravated HFD-induced microglia activation and inflammatory reactions in the ARC region of the hypothalamus.Considering that Sirt6 could reduce inflammation and protect microglia,Sirt6 can be regarded as a protector to prevent microglia from HFD-induced injuries.Therefore,Sirt6 in microglia may be an important therapeutic target for obesity.(2)DSS alleviated obesity induced by HFD in mice.DSS reduced the body mass and adipose tissue weight of obese mice induced by HFD and improved the blood lipid status of these mice.Moreover,DSS alleviated the pathological injuries related to obesity induced by HFD,decreased glucose tolerance impairment,reduced insulin resistance,and mitigated hepatic gluconeogenesis in mice.Besides,DSS up-regulated the expression of Sirt6 in microglia in the ARC region of the hypothalamus induced by HFD and mitigated the inflammatory reaction in the hypothalamus of obese mice.Based on the protective effect of Sirt6 in microglia on diet-induced obesity,it can be optimistically maintained that DSS upregulated the expression of Sirt6 in the microglia of mice.This finding is of vital importance to diet-induced obesity.DSS may improve diet-induced obesity by improving the activation of hypothalamic microglia and reducing hypothalamic inflammatory responses. |
PDF Full Text Request