| Background and aims:Diabetes is a group of metabolic diseases characterized by a continuous increase of blood glucose level,which can be clinically divided into type ? diabetes mellitus(T1DM)and type ? diabetes mellitus(T2DM)according to its dependence on insulin.Multiple organ damages such as diabetic foot,eye disease,kidney disease and cardiomyopathy may occur in the late stage of diabetes.The treatment of diabetes depends on its types and whether it is accompanied by complications.In general,type? diabetes requires long-term insulin therapy,while type ? diabetes treatment includes the basis of diet adjustment,as well as using drugs that promoting insulin secretion,enhancing insulin sensitivity or delaying food absorption.In severe cases,insulin therapy can also be introduced.Therefore,the development of new drugs for the effective treatment of diabetes is of great significance.BefA(Beta cell expansion factor A)is a protein produced by human intestinal flora.Studies have shown that this protein promoted the proliferation of pancreatic ?cells,suggesting that it may have a potential value for the treatment of diabetes.We first constructed a prokaryotic BefA expression vector,using E.coli BL21 expression system for expression and purification to obtain higher purity of BefA protein,then we evaluated its treatment effect by intragastric administration on type ? and type ?diabetes mouse models.The results showed that oral administration of BefA protein could reduce blood glucose in diabetic mice,inhibit tissue inflammation,promote islet ? cell proliferation and insulin secretion in a concentration-dependent manner,and at the same time it produced beneficial regulatory effects on the intestinal flora of diabetic mice.Experimental methods:1.BefA protein expression and purification:The artificially synthesized BefA sequence was inserted into the prokaryotic expression vector pET-28C,and then transferred to the BL21 strain.Protein expression and purification were induced by IPTG and His-tag nickel beads respectively.The purity and bands of BefA protein were detected by SDS-PAGE electrophoresis and Western-blot,and the purified protein concentration was determined by BCA protein concentration kit.2.Preparations of diabetic mouse models,the administration of BefA and the evaluation of efficacy1)Preparation of type ? diabetic mouse model:Mice were injected intraperitoneally with STZ(50 mg/kg/d)for five consecutive days.When the blood glucose concentration rose to 11.1 mM or above and stabilized for seven days,we considered it as successful modeling.The control group was injected with equal volume of normal saline at the same time.Sixty eight-week-old male C57BL/6 mice were divided into 4 groups of 15 mice each:(1)normal control group(C group);(2)TIDM model group(M group);(3)low concentration BefA protein treatment group(MB 10 group):10 ?g/BefA/mouse/2 days;(4)high concentration BefA protein treatment group(MB 50 group):50?g/BefA/mouse/2 days.2)Preparation of type 2 diabetic mouse model:The experimental mice were fed with high fat diet(HFD)for six weeks combined with low-dose of STZ(30 mg/kg)intraperitoneal injection to induce type ? diabetic mouse model.When the blood glucose concentration was 11.1 mM or above and stabilized for 7 days,it is regarded as successful modeling.A total of 75 male C57BL/6 mice at 8 weeks of age were divided into 5 groups and 15 mice in each group:(1)normal control group(C group);(2)T2DM model group(M group);(3)low concentration BefA treatment group(MB 5 group):5 ?g BefA/mouse/2 days;(4)medium concentration BefA treatment group(MB 20 group):20 ?g BefA/mouse/2 days;(5)high concentration BefA treatment group(MB 40 group):40 ?g BefA/mouse/2 days.3)Administration of BefA and the evaluation of the efficacy:BefA protein was resuspended in a coating solution(0.01%gelatin prepared with 0.9%physiological saline)and administered intragastrically every other day for 14 times.The therapeutic effects of BefA on diabetic mouse models were evaluated by measuring blood glucose,body weight and glucose tolerance assay.4)Analysis of the mechanisms:Western-blot was used to detect the expression levels of pancreatic inflammation signaling pathway proteins(TLR-4,NF-?B)and apoptosis proliferation pathway proteins(Bax,Bcl-2,PDX-1).q-PCR was used to detect the transcription of pancreatic inflammatory factors(TNF-?,IL-1?).Immunohistochemistry,HE staining and immunofluorescence techniques were used to evaluate pancreatic islet tissue damage,pancreatic ? cell proliferation and insulin secretion.The effect of BefA protein on the number and species of gut microbiota was analyzed by high-throughput sequencing in a mouse model of type ? diabetes.The oil red O staining techniques was used to detect liver cell morphology and oil droplet size in type ? diabetic mouse model.In addition,the expression and transcription of CEBP-? were also detected in liver to evaluate the effect of BefA proteins on lipid metabolism in T2DM mice.Experimental results:1.BefA protein expression and purification:The results showed that the pET-28C-BefA expression vector was successfully constructed.SDS-PAGE electrophoresis and Western-blot showed that the purity of the purified BefA protein was between 85-90%.2.Therapeutic effects of BefA on type ? diabetic mouse model:(1)Oral administration of BefA protein can significantly reduce blood glucose level and relieve weight loss symptoms of type ? diabetic mice in a concentration-dependent manner;(2)BefA protein treatment can reduce the expression levels of TLR-4 and NF-?B as well as down-regulate the mRNA levels of IL-1? and TNF-? in pancreas in a concentration-dependent manner;(3)BefA protein can down-regulate the ratio of Bax/Bcl-2 protein expression and increase PDX-1 expression and transcription level in pancreas in a concentration-dependent manner;BefA protein can restore pancreatic tissue morphology,promote the proliferation of islet ? cells and increase insulin secretion level;(4)High-throughput sequencing results of intestinal flora showed that high concentration of BefA treatment(MB 50 group)increased the abundance of intestinal probiotic lactic acid bacteria at the level of family(f),genus(g)and order(o),and made the structure of intestinal flora closer to the normal control group.3.Therapeutic effects of BefA on type ? diabetic mouse model:(1)Oral administration of BefA reduced hyperglycemia and weight increase symptom in T2DM mice in a concentration-dependent manner;(2)BefA could reduce the expression levels of inflammatory factors(TLR-4,NF-?B,IL-1? and TNF-?)in pancreas and liver in a concentration-dependent manner;(3)BefA down-regulated the protein expression ratio of Bax/Bcl-2,increased the expression of PDX-1,promoted the proliferation of ? cells and increased the level of insulin secretion in the pancreas of T2DM mouse model;(4)BefA down-regulated the expression and transcription levels of CEBP-? in the liver of T2DM mouse model in a concentration-dependent manner,suggesting that the protein might inhibit the formation of fat in the diabetic mouse model;(5)Low concentration of BefA has no significant therapeutic effect on T2DM mouse model.Conclusions:BefA efficiently relieved the increase of blood glucose levels in both ? and ?diabetic mice,and inhibited the bodyweight loss in type ? diabetic mice or the increase of the bodyweight in type ? diabetic mice and its mechanism may be related to its inhibition of inflammatory response and apoptosis in pancreatic tissue,promotion of islet ? cell differentiation and proliferation,and insulin secretion.Moreover,the corrective effect of BefA on intestinal flora disorder in diabetic mice is also beneficial to the treatment of diabetes,and its inhibition of triglyceride synthesis in liver cells is also beneficial to the treatment of type ? diabetes. |
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