Effect of berberine on hyperglycaemia and gut microbiota composition in type 2 diabetic Goto-Kakizaki rats

BACKGROUND A recent investigation showed that the prevalence of type 2 diabetes mellitus(T2DM)is 12.8%among individuals of Han ethnicity.Gut microbiota has been reported to play a central role in T2DM.Goto-Kakizaki(GK)rats show differences in gut microbiota compared to non-diabetic rats.Previous studies have indicated that berberine could be successfully used to manage T2DM.We sought to understand its hypoglycaemic effect and role in the regulation of the gut microbiota.AIM To determine whether berberine can regulate glucose metabolism in GK rats via the gut microbiota.METHODS GK rats were acclimatized for 1 wk.The GK rats were randomly divided into three groups and administered saline(Mo),metformin(Me),or berberine(Be).The observation time was 8 wk,and weight,fasting blood glucose(FBG),insulin,and glucagon-like peptide-1(GLP-1)were measured.Pancreatic tissue was observed for pathological changes.Additionally,we sequenced the 16S rRNA V3-V4 region of the gut microbiota and analysed the structure.RESULTS Compared with the Mo group,the Me and Be groups displayed significant differences in FBG(P<0.01)and GLP-1(P<0.05).A significant decrease in weight and homeostatic model assessment-insulin resistance was noted in the Be group compared with those in the Me group(P<0.01).The pancreatic islets of the Me-and Be-treated rats showed improvement in number,shape,and necrosis compared with those of Mo-treated rats.A total of 580 operational taxonomic units were obtained in the three groups.Compared to the Mo group,the Me and Be groups showed a shift in the structure of the gut microbiota.Correlation analysis indicated that FBG was strongly positively correlated with Clostridia_UCG-014(P<0.01)and negatively correlated with Allobaculum(P<0.01).Body weight showed a positive correlation with Desulfovibrionaceae(P<0.01)and a negative correlation with Akkermansia(P<0.01).Importantly,our results demonstrated that Me and Be could significantly decrease Bacteroidetes(P<0.01)and the Bacteroidetes/Firmicutes ratio(P<0.01).Furthermore,Muribaculaceae(P<0.01;P<0.05)was significantly decreased in the Me and Be groups,and Allobaculum(P<0.01)was significantly increased.CONCLUSION Berberine has a substantial effect in improving metabolic parameters and modulating the gut microbiota composition in T2DM rats.

Characterization of gut microbial and metabolite alterations in faeces of Goto Kakizaki rats using metagenomic and untargeted metabolomic approach

BACKGROUND In recent years,the incidence of type 2 diabetes(T2DM)has shown a rapid growth trend.Goto Kakizaki(GK)rats are a valuable model for the study of T2DM and share common glucose metabolism features with human T2DM patients.A series of studies have indicated that T2DM is associated with the gut microbiota composition and gut metabolites.We aimed to systematically characterize the faecal gut microbes and metabolites of GK rats and analyse the relationship between glucose and insulin resistance.AIM To evaluate the gut microbial and metabolite alterations in GK rat faeces based on metagenomics and untargeted metabolomics.METHODS Ten GK rats(model group)and Wistar rats(control group)were observed for 10 wk,and various glucose-related indexes,mainly including weight,fasting blood glucose(FBG)and insulin levels,homeostasis model assessment of insulin resistance(HOMA-IR)and homeostasis model assessment ofβcell(HOMA-β)were assessed.The faecal gut microbiota was sequenced by metagenomics,and faecal metabolites were analysed by untargeted metabolomics.Multiple metabolic pathways were evaluated based on the differential metabolites identified,and the correlations between blood glucose and the gut microbiota and metabolites were analysed.RESULTS The model group displayed significant differences in weight,FBG and insulin levels,HOMA-IR and HOMA-βindexes(P<0.05,P<0.01)and a shift in the gut microbiota structure compared with the control group.The results demonstrated significantly decreased abundances of Prevotella sp.CAG:604 and Lactobacillus murinus(P<0.05)and a significantly increased abundance of Allobaculum stercoricanis(P<0.01)in the model group.A correlation analysis indicated that FBG and HOMA-IR were positively correlated with Allobaculum stercoricanis and negatively correlated with Lactobacillus murinus.An orthogonal partial least squares discriminant analysis suggested that the faecal metabolic profiles differed between the model and control groups.Fourteen potential metabolic biomarkers,including glycochenodeoxycholic acid,uric acid,13(S)-hydroxyoctadecadienoic acid(HODE),N-acetylaspartate,β-sitostenone,sphinganine,4-pyridoxic acid,and linoleic acid,were identified.Moreover,FBG and HOMA-IR were found to be positively correlated with glutathione,13(S)-HODE,uric acid,4-pyridoxic acid and allantoic acid and negatively correlated with 3-α,7-α,chenodeoxycholic acid glycine conjugate and 26-trihydroxy-5-β-cholestane(P<0.05,P<0.01).Allobaculum stercoricanis was positively correlated with linoleic acid and sphinganine(P<0.01),and 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate was negatively associated with Prevotella sp.CAG:604(P<0.01).The metabolic pathways showing the largest differences were arginine biosynthesis;primary bile acid biosynthesis;purine metabolism;linoleic acid metabolism;alanine,aspartate and glutamate metabolism;and nitrogen metabolism.CONCLUSION Metagenomics and untargeted metabolomics indicated that disordered compositions of gut microbes and metabolites may be common defects in GK rats.

Dan-gua Fang (丹瓜方) Improves Glycolipid Metabolic Disorders by Promoting Hepatic Adenosine 5'-monophosphate Activated Protein Kinase Expression in Diabetic Goto-kakizaki Rats

Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.Methods：Forty 13-week-old diabetic Goto-Kakizaki（GK） rats were randomly divided into model,Dan-gua Fang,metformin and simvastatin groups（n=10 for each）,and fed high-fat diet ad libitum.Ten Wistar rats were used as normal group and fed normal diet.After 24 weeks,liver expression of AMPK α mRNA was assessed by real-time PCR.AMPK α and phospho-AMPK α protein expression in liver was evaluated by Western blot.Liver histomorphology was carried out after hematoxylin-eosin staining,and blood glucose（BG）,glycosylated hemoglobin A1c（HbA1c）,food intake and body weight recorded.Results：Similar AMPK α mRNA levels were found in the Dan-gua Fang group and normal group,slightly higher than the values obtained for the remaining groups（P〈0.05）.AMPK α protein expression in the Dan-gua Fang group animals was similar to other diabetic rats,whereas phospho-AMPK α（Thr-172） protein levels were markedly higher than in the metformin group and simvastatin group（P〈0.05）,respectively.However,phosphor-AMPKa/AMPK α ratios were similar in all groups.Dan-gua Fang reduced fasting blood glucose with similar strength to metformin,and was superior in reducing cholesterol,triglycerides,high-density lipoprotein cholesterol as well as improving low-density lipoprotein cholesterol in comparison with simvastatin and metformin.Dan-gua Fang decreases plasma alanine aminotransferase（ALT） significantly.Conclusion：Dan-gua Fang,while treating phlegm-stasis,could decrease BG and lipid in type 2 diabetic GK rats fed with high-fat diet,and effectively protect liver histomorphology and function.This may be partly explained by increased AMPK expression in liver.Therefore,Dan-gua Fang might be an ideal drug for comprehensive Intervention for glucose and lipid metabolism disorders in type 2 diabetes mellitus.

Asiatic acid mitigates hyperglycemia and reduces islet fibrosis in Goto-Kakizaki rat,a spontaneous type 2 diabetic animal model

The Goto-Kakizaki(GK) rat is a spontaneous type 2 diabetic animal model,which is characterized by a progressive loss of beta islet cells with fibrosis.In the present study,the hypoglycemic effect of asiatic acid(AA) in GK rats was examined.GK rats receiving AA at a daily dose of 25 mg·kg-1 for four weeks showed a significant reduction in blood glucose levels.Age-matched normal Wistar rats were given 0.5% sodium carboxymethyl cellulose(CMC-Na) solution for the same periods and used as control.Compared to the normal Wistar rats,GK rats treated with AA showed improvement in insulin resistance partially through decreasing glucose level(P < 0.01) and insulin level(P < 0.05).Furthermore,the results of immunohistochemistry indicate that AA treatment reduced islet fibrosis in GK rats.Fibronectin,a key protein related to islet fibrosis,was over-expressed in GK rats,which was reversed significantly by AA treatment(P < 0.05).These findings suggest that AA has a beneficial effect on lowering blood glucose levels in GK rats and improves fibrosis of islets in diabetes,which may play a role in the prevention of islets

黄连解毒汤对自发性糖尿病大鼠免疫炎症因子及损伤的影响

目的:观察黄连解毒汤对自发性糖尿病大鼠心肌免疫炎症因子的影响及对心肌损伤的防治作用。方法:GK大鼠30只随机分为GK对照组、二甲双胍组、黄连解毒汤组,正常大鼠10只作为正常对照组分别灌胃12周,测定心肌匀浆核因子-κB(NF-κB)、细胞间黏附分子-1(sICAM-1)、白介素-1(IL-1)、肿瘤坏死因子(TNF-α)、内皮素-1(ET-1)含量。取心肌切片作HE染色。结果:2药物组血糖均下降(P<0.01)。GK对照组各炎症因子升高(P<0.01～0.05),黄连解毒汤组降低(P<0.01～0.05),其中NF-κB、TNF-α、ET-1与二甲双胍比较下降程度较大(P<0.01～0.05)。HE染色显示黄连解毒汤组心肌损伤明显减轻。结论:黄连解毒汤可防治糖尿病心肌炎症损伤。

黄芪调节自发糖尿病肾病鼠层粘连蛋白表达的实验研究

目的:观察黄芪对自发糖尿病肾病大鼠肾小球层粘连蛋白表达的影响。方法:将实验用6月龄SPF级GK大鼠和Wistar大鼠随机分为正常对照组、糖尿病组、黄芪治疗组。治疗16周,观察治疗后大鼠的尿素氮、血肌苷,内生肌苷清除率、24小时尿蛋白排泄率,免疫组化检测肾组织层粘连蛋白表达。结果:造模组大鼠均出现肾脏功能有损害。黄芪能改善自发糖尿病肾病大鼠基本状况,降低糖尿病大鼠的尿素氮、血肌苷、24h尿白蛋白排泄率,增加内生肌苷清除率,层粘连蛋白表达显著下调。结论:黄芪可通过降低层粘连蛋白的表达,对肾脏起保护作用。

通心络胶囊对自发性糖尿病模型大鼠心肌微血管炎症损伤及血管痉挛的防治作用

目的:观察通心络胶囊对自发性糖尿病模型大鼠心肌微血管炎症损伤及血管痉挛的防治作用。方法:4月龄GK大鼠30只,随机分为GK对照组、通心络高低剂量组。另取4月龄Wister大鼠10只,作为正常对照组。各组分别灌胃12周,取心肌切片作HE染色、NF-κB免疫组化染色。取心肌匀浆,酶联免疫方法测定心肌NF-κB、细胞间黏附分子-1(sICAM-1)、白介素-1(IL-1)含量,放免法测定肿瘤坏死因子(TNF-α)、内皮素-1(ET-1)、血管紧张素-Ⅱ(AT-Ⅱ)含量。结果:①与正常大鼠比较,GK大鼠心肌NF-κB阳性表达增高(P<0.01),通心络组表达降低(P<0.01),匀浆NF-κB、sICAM-1、TNF-α、ET-1含量均有不同程度增高(P<0.01～0.05),通心络组降低(P<0.01～0.05),在降低TNF-α、ET-1方面,高低剂量通心络有显著性差异(P<0.01～0.05)。②IL-1、AT-Ⅱ、NO含量,GK大鼠与正常大鼠无差异(P>0.05),通心络组却能够升高IL-1、AT-Ⅱ含量(P<0.01)。结论:通心络胶囊能抑制心肌组织NF-κB阳性表达,降低心肌匀浆NF-κB、sICAM-1、TNF-α等炎症因子含量,降低ET-1含量,减轻心肌病变。对IL-1、AT-Ⅱ、NO无明显影响。对微血管痉挛有一定的防治作用。

自发性2型糖尿病动物模型GK大鼠的发病特征研究

目的:研究自发性2型糖尿病动物模型(GK)大鼠的发病特征。方法:以Wistar大鼠(n=12)为对照,从第13周龄起连续监测GK大鼠(n=61)的基础血糖值、体重、摄食量和饮水量。第32周龄时,开展糖耐量试验,进行大便常规和肝肾指数检查,检测血浆中血脂、肾功能和糖化血红蛋白(HbA1c)的水平,取胰腺和肾脏进行病理检查。结果:GK大鼠在第24周龄时开始发病,血糖稳定地维持在11.0 mmol.L-1以上的高血糖水平,HbA1c水平亦明显升高,至少可以维持到第32周龄;期间还出现类似于糖尿病病人"消瘦"、"多食"、"多饮"等临床症状;死亡率相对较低,仅为11.47%左右。GK大鼠对糖负荷的代谢能力降低,胰腺和肝脏组织病理改变明显加重。结论:GK大鼠在第24～32周龄期间发病,具有明显的、稳定的糖尿病发病特征,且死亡率低,有利于开展糖尿病的发病机制和药物的有效性评价研究。

川芎嗪调节自发糖尿病肾病鼠Ⅲ型前胶原表达的实验研究

目的:观察川芎嗪对自发糖尿病肾病大鼠肾小球Ⅲ型前胶原基因表达的调节。方法:将实验用6月龄SPF级GK大鼠和Wistar大鼠随机分为正常对照组、糖尿病组、川芎嗪治疗组。治疗16周。观察治疗后大鼠的尿素氮、血肌苷,内生肌苷清除率、24小时尿蛋白排泄率,免疫组化检测肾组织Ⅲ型前胶原表达。结果:①造模组大鼠均出现肾脏功能有损害②川芎嗪能改善自发糖尿病肾病大鼠基本状况,降低糖尿病大鼠的尿素氮、血肌苷、24h尿白蛋白排泄率,增加内生肌苷清除率,Ⅲ型前胶原表达显著下调。结论:川芎嗪可通过降低Ⅲ型前胶原的表达,对肾脏起保护作用。

桂枝汤对自发性糖尿病大鼠炎症因子及心肌损伤的影响

目的:观察桂枝汤对自发性糖尿病大鼠(Goto-Kakizaki diabetes prone rat,GK大鼠)核因子-κB(NF-κB)、细胞间黏附分子-1(sICAM-1)、白介素-1(IL-1)、肿瘤坏死因子(TNF-α)、内皮素-1(ET-1)的影响,及对心肌损伤的防治作用。方法:GK大鼠30只,随机分为GK对照组、二甲双胍组、桂枝汤组,另取正常大鼠10只作为正常对照组,分别ig 12周,测定各组心肌匀浆NF-κB,sICAM-1,IL-1,TNF-α,ET-1含量。取心肌切片作HE染色。结果:与GK对照组比较二甲双胍组血糖下降(P<0.01),桂枝汤组轻度升高(P>0.05)。GK对照组NF-κB,sICAM-1,IL-1,TNF-α,ET-1各炎症因子较正常大鼠明显升高(P<0.01或P<0.05)。二甲双胍组IL-1,TNF-α降低(P<0.01或P<0.05),桂枝汤组NF-κB,IL-1,TNF-α,ET-1明显降低(P<0.01～0.05),并且比二甲双胍降低程度大,其中IL-1,TNF-α具有显著性差异(P<0.01或P<0.05)。HE染色染色示桂枝汤组心肌损伤明显减轻。结论:桂枝汤可减少炎症因子蓄积,防治糖尿病心肌损伤。