The Mechanism Of Mulberry Leaf Effective Extracts In Ameliorating KKA~y Mice Insulin Resistance Via NF-κB

BackgroundMore than 150 million people with type 2 diabetes are suffering distress, and this figure is expected to be double in 2020. Most patients are obese with type 2 diabetes, often associated with hyperlipidemia, which are prone to severe complications such as kidney failure, blindness, slow wound healing, artery disease (including coronary artery disease), if improper control. Many scholars believe that type 2 diabetes may be caused by an autoimmune disease mediated by inflammatory cytokines, and this view was called "inflammatory theory" of type 2 diabetes. Inflammatory mechanisms of type 2 diabetes has since become a hot spots which diabetes scholars researched.Studies have shown that excessive secretion of inflammatory cytokines and the state of chronic inflammation caused by sustained activation of inflammatory pathways, may induce insulin resistance and diabetes type 2 diabetes in animals and patients. Nuclear factor KappaB (NF-κB) is transcription factors widely distributing in many cell types, and plays an important role in signaling transmission and gene expression in the inflammatory reaction. IκBα is the inhibitor of NF-κB, and it can control the immune and inflammatory responses. Due to the presence of IκBα, NF-κB is in a stationary state in the cytoplasm. Many outer cellular immune stimulant can activate IκKβ by receptor-mediated pathways, which lead to the phosphorylation and degradation of IκBα, resulting in activation of NF-κB. Then NF-κB is into the nucleus and promote the release of pro-inflammatory cytokines. Overexpression of proinflammatory cytokines is an important factor leading to insulin resistance. Researching the inhibition of Mulberry leaf on NF-κB activity and expression of inflammatory cytokines insulin-induced HepG2 cells and spontaneous type 2 diabetic mice KKAy may have important clinical significance in the course of treatment of type 2 diabetes, but the mechanism has yet to be deeper research.Currently, mainly agents for the treatment of type 2 diabetes are into insulin and oral hypoglycemic agents, oral hypoglycemic agents would be divided into as insulin secretagogues, insulin sensitizers, α-glucosidase inhibitor and biguanide hypoglycemic traditional medicine; with the development of some new hypoglycemic drugs has gradually into clinical use, such as GLP-1 agonists---Ai game and Delilah PUTAI, DPP-4 inhibitor---sitagliptin and vildagliptin, SGLT-2 inhibitors and GPR119 agonists so on. These new drugs were only by a few thousand patients, may mask a lot of cases.It would have yet to accumulate clinical data for their practical efficacy, safety and side effects, so far hypoglycemic agents were still majorly for the traditional clinical, but western medicine although from the surface fast lower blood sugar, but not fundamentally reduce the occurrence of diabetes, but there are many side effects, so look for hypoglycemic Chinese medicine has become a new therapeutic strategies. Traditional Chinese medicine have a lot of practice in the treatment of type 2 diabetes and its complications and accumulated a great deal of clinical experience. Traditional Chinese medicine has been widely used for the treatment of type 2 diabetes. Mulberry leaves are dried leaves of Sankoh plant, belong to Ganhan goods, can lower blood pressure, blood lipids, anti-inflammatory, has been widely used for the treatment of Diabetes in the folk. Our group has been working on Mulberry hypoglycemic, from raw material selection, mulberry leaf extract effective extraction and separation and purification thereof, to hypoglycemic mechanism, a series of studies.In view of the above analysis, the subject studied HepG2 cells and spontaneous type 2 diabetic KKAy mice to observe the role of Mulberry alkaloid (MLE), Mulberry leaves polysaccharides (MLP), Mulberry leaves flavonoids (MLF) on IκKβ/NF-κB signaling pathway related gene protein inhibition and expression of inflammatory cytokines in insulin-induced HepG2 cells and spontaneous type 2 diabetic mice KKAy, and to explore protective effect of Mulberry leaf effective parts on type 2 diabetes in order to provide theoretical and experimental evidence for its clinical application. As follows:Chart 1 Effects of Mulberry leaves extracts on IκKβ/NF-κB signaling pathway related gene expression in IR-HepG2 cellsObjective Establishing insulin resistance model in HepG2 cells to explore the effects of Mulberry leaves effective extracts on mRNA expression of IκKβ, IκBα gene and NF-κB activation in the model cell.Method To cultivate HepG2 cells with 10μg·mL-1 insulin culture medium for 48 h to establish IR-HepG2 model. The model cells were randomly divided into model group, polysaccharides group, flavonoids group, alkaloids group, rosiglitazone group, and for HepG2 cells as the control group.Detecting the proliferation of HepG2 cells through cck8 assay, glucose oxidase-peroxidase (GOD-POD) assay was used to detect residual glucose in the culture medium to determine the optimal intervention concentration of each experimental group. Q-PCR was used to mRNA expression of IκKβ and IκBα.In addition,Western Blot was used to expression of IκBα to reflect NF-κB activity. Results After high insulin-induced insulin resistance in HepG2 cells,the expressi of IκKβ and NF-κB activity was significantly enhanced, the degradation of NF-κB binding protein IκBα was also obvious; After Mulberry leaf extracts intervention, the expression of IκKβ and NF-κB activity was significantly reduced, degradation of IκBα was suppressed.Conclusion After insulin-induced insulin resistance in HepG2 cells, NF-κB pathway was activated in a certain extent, which indicated the presence of a certain relationship between insulin resistance and NF-κB inflammation pathway; Mulberry leaf extract can inhibit NF-κB activation, then will provides a theoretical basis for the prevention and treatment of type 2 diabetes.Chart 2 Effects of mulberry leaves extract on glucose metabolism in spontaneous type 2 diabetes in KKAy miceObjective To investigate the effects of mulberry leaves extract (polysaccharides, flavonoids, alkaloids) on spontaneous type 2 diabetes KKAy mice.Method The KKAy mice were randomized divided into five groups, the model group (IR), mulberry polysaccharides (MLP,1000 mg·kg-1), mulberry flavonoids (MLF,1000 mg·kg-1), mulberry alkaloid (MLA,30 mg·kg-1) and rosiglitazone (RSG,2 mg·kg-1), eight in each group, and the other to 10 C57BL/6 mice were as normal control group (NC). The kkay mice were fed a special high-fat diet while the normal control group were fed a normal diet.The experimental groups were administrated by gavages (i.g.) to diabetes for 8 consecutive weeks, model group and the normal group received the same volume of distilled water.The fasted blood sample was collected from the tail for blood glucose measurement every 1 weeks, with a portable glucometer. Animal was gavages (i.g.) according to weight which was measured every 1 week. After the last administration for 1h, anesthetized by chloral hydrate, opened the abdominal cavity, quickly stripped liver, epididymal fat, weighed to calculate the coefficient of liver and epididymal fat, lastly placed in liquid nitrogen.Results Mulberry leaves extract (polysaccharides, flavonoids, alkaloids) were able to reduce the weight of KKAy mice, liver and epididymal fat index coefficient, lowered epididymal fat weight, improved fasting glucose levels, increased liver glycogen in KKAy mice, had statistically significant difference compared with model group (P<0.05).Conclusion Mulberry leaves extract (polysaccharides, flavonoids, alkaloids) reduced the weight in spontaneous type 2 diabetes KKAy mice, lowered fasting blood sugar and increased liver glycogen in favor of long-term stability on blood sugar control,while inhibited liver, adipose tissue swelling due to the high sugar in mice.Chart 3 Influence of Mulberry leaf extract on serum factor in spontaneous type 2 diabetic KKAy miceObjective To investigate impact of Mulberry leaf extract (polysaccharides, flavonoids, alkaloids) on serum IL-6, hs-CRP, and TNF-a in spontaneous type 2 diabetes KKAy mice.Method The KKAy mice were randomized divided into five groups, the model group (IR), mulberry polysaccharides (MLP,1000 mg·kg-1), mulberry flavonoids (MLF,1000 mg·kg-1), mulberry alkaloid (MLA,30 mg·kg-1) and rosiglitazone (RSG,2 mg·kg-1), eight in each group, and the other to 10 C57BL/6 mice were as normal control group (NC). The kkay mice were fed a special high-fat diet while the normal control group were fed a normal diet.The experimental groups were administrated by gavages (i.g) to diabetes for 8 consecutive weeks, model group and the normal group received the same volume of distilled water. After the last administration for 1h, anesthetized by chloral hydrate, decapitated for blood, then centrifuged for 15min in 3000r/min for the serum, stored in-70 "C. According to the ELISA kit for detection.Results Compared with the normal control C57BL/6 mice, the levels of serum CRP,TNF-a and IL-6 were significantly increased in model group(P<0.01); after intervention of Mulberry leaves extracts for four weeks,the levels of serum IL-6, hs-CRP, and TNF-a were significantly decreased in each treatment group, compared to model group(P<0.05).Conclusion Mulberry leaves reduce insulin resistance in KKAy mice to restore normal metabolism through regulating the levels of serum IL-6, CRP, and TNF-a.Chart 4 Effects of Mulberry leaf extract the expression of protein IκKβ, p-IκBα of liver and epididymal adipose tissue in spontaneous type 2 diabetes KKAy miceObjective To investigate effects of Mulberry leaf extract the expression of protein IκKβ, p-IκBα of liver and epididymal adipose tissue in spontaneous type 2 diabetes KKAy mice.Method Putting from the liquid nitrogen method of liver and epididymal adipose tissue to placed in a mortar, then grounded to a powder in liquid nitrogen. The mixture of protease inhibitors tissue lysate and milling was added then continue to ground until the homogenate, centrifugeed for the protein solution. The protein concentration was detected by BCA assay and expression of protein IκKβ, p-IκBα through Western Blot assay.Results Compared with normal control group (NC), the expression of protein IκKβ, p-IκBα in liver and epididymis tissue was elevated in model group; after drug intervention, compared with the model group, the protein IκKβ, p-IκB a expression in liver and epididymal tissue of experimental group was significantly decreased (P<0.05), IκBα degradation was significantly inhibited.Conclusion The IκKβ/NF-κB inflammation signaling pathway was activated in liver and adipose tissue of spontaneous type 2 diabetes KKAy mice, levels of IκBα phosphorylation proteins IκKβ were elevated.Mulberry leaf extracts (polysaccharides, flavonoids, alkaloids) can significantly reduced IκBα phosphorylation and protein IκKβ levels in liver and epididymal adipose tissue.