Theoretical And Experimental Study Of Diabetes (Type 2 Diabetes) Treated From "Spleen"

Type 2 Diabetes mellitus (T2DM) is a metabolic disease caused by a variety of causes and characterized by chronic hyperglycemia,belongs to the category of "XiaoKe" of traditional Chinese medicine. Modern physicians Zhang Xi-chun proposed "Xiaoke commence in middle jiao, and develop to the upper and lower", reveals the position, nature and trend of T2DM. Therefore, this study puts forward that the T2DM is closely related to "spleen", and points out T2DM is mainly due to subtle distribution and metabolic disorder and imbalance caused by dysfunction of spleen in transportation. At the same time,modern medicine considers that T2DM are with abnormal metabolism of sugar, fat and protein caused byinsulin secretion or action defects, islet beta cell function and insulin resistance are the two pathophysiological basis of T2DM.Objective:This study puts forward a academic viewpoint "XiaoKe treaded from the spleen" in TCM. Use "JINLIDA granule (JLD)"which guided from "activating spleen essence" treatment.The T2DM rat models were developed to observe diabetic rats islet function and injury in islet cells and the intervention effects of JLD. The Insulin resistance rat models were developed to observeglucolipid metabolism and insulin sensitivity and the intervention effects of JLD. Additionally, the Palmitic acidwas used to induce the damages in islet β cells. Then the effects and mechanism of JLD were explored on the model in vitro. Through the above experiments the promising treatment methods maybe supplied for clinic,and these data would give scientific explanation for the theory of treating diabetes (T2DM) from the"spleen".Methods:This research is divided into the following two parts:Theoretical research methods:Carding the acknowledgement of etiology, pathogenesis, treatmentof the traditional understanding of XiaoKe"(T2DM). Analysis the theoryputed from the modern physicians Zhang Xichun that "commence in middle jiao, and develop to the upper and lower" in theory and clinical, application from "spleen" theory, put forward " subtle distribution and metabolic disorder and imbalance caused by dysfunction of spleen" is the main pathogenesis of T2DM, "phlegm and blood stasis block blood vessels caused by dysfunction of spleen " is the root cause of T2DM complications, and establish "activating spleen essence" therapeutic principle and synthesize some methods to cure spleen,that is:raise spleen Yin, dry spleen dampness, drain spleen heat, warm the spleen Yang, chang temper, form JLD particle, and analysis of JLD particles formula characteristics and clinical and experimental study progress, so as to provide effective treatment for T2DM.Experimental research methods:1. Intervention study of Jinlida in T2DM rats Islet function injury90 male SD rats were randomly divided into normal group, pioglitazone group(4.5mg/kg body weight),model group,JLD low,middle,high doses group,(0.75,1.5,3. Omg/kg body weight).15 mice in each group.Except the normal group, the rest of groups were fed with high-fat food.6 weeks later,15mg/kg streptozotocin was injected intraperitoneally to develop T2DM model rats. After 72h, the rats fasting-glucose exceeding 13.9 mmol/L were adopted to feed high-fat food and and corresponding drug respectively for 6 weeks. Fasting 12 h after the end treatment, then the blood serum was separatedto detect of index including blood glucose(FBG) and insulin (FINS)of each group were measured to calculate index of β-cell function in HOMA(HOMA-β). Organelles injury of islet β-cells were investigated by transmission electron microscopy.Casepase3、p62、Atg7、LC3Ⅰ、LC3Ⅱ protein were detected by Western blotting.2. Intervention study of Jinlidain IR rat islet glucolipid metabolism and insulin sensitivity.60 male SD rats were randomly divided into normal group, pioglitazone group(4.5mg/kg body weight),model group, JLD low,middle,high doses group,(0.75,1.5,3.0mg/kg body weight).10 mice in each group.Except the normal group, the rest of groups were fed with high-fat food.6 weeks later, Choose the model of animals by the oral glucose tolerance test, the rats fasting-glucose belowing 13.9 mmol/L and AUC area has statistically significant with normal group were adopted to feed high-fat food and and corresponding drug respectively for 6 weeks.Fasting 12 h after the end treatment, flood insulin (FINS)of each group were measured to calculate index of IRby HOMA(HOMA-IR).Oil red O staining was used toobserve the accumulation of fatof rats liver cells.Liver cell ultrastructurewere observed by transmission electron microscopy.Resistin、IL-6、TNFawere detected by enzyme-linked immunosorbent assay.p-AMPK、AMPK、p-ACC、ACC、p-PI3K、PI3K、p-Akt、Akt、GLUT4were detected by WesternBlot.3. Intervention study of Jinlida inthe Palmitic acidinduced the injury in islet β cells.INS-1 cells were cultured in vitro, cell survival activity were detecked by CCK-8 method to determinedthe The toxicity of JLD (100,200,300,400 μg/mL).After giving JLD and PA, CCK-8 method was used to detect cell survival activity, RT-PCR method were detected cell BAX, Bcl-2, Atg7, Beclin-1 gene expression.. Compound C was used to treat the cells in order to study its influence on the expression of p62.1. Intervention study of Jinlida in T2DM ratslslet function injury:Compared with control group, fasting glucose and insulin in STZ group were increased significantly (P< 0.01). TEM indicated the nuclear chromatin condensed, volume were reduced. HOMA-β was reduced. LC3Ⅱ、Atg7protein was significantly decreased、caspase3、p62、LC3I protein was significantly increased. Compared with Model group,the mice from JLD group showed ameliorated islet P-cells changes.ILD reduced casepase3、p62、LC3I protein expressionraised HOMA-βand Atg7、 LC3Ⅱ、Atg7protein expression.(P< 0.05, P< 0.01).2. The result of Intervention study of Jinlidain IR rat islet glucolipid metabolism and insulin sensitivity:Compared with control group, the weight、FBG and cholesterol (TC)、triglyceride (TG)、low-density lipoprotein (LDL-C) of HF group were increased significantly,while high-density lipoprotein (HDL-C) decreased significantly(P<0.05).Compared with HF group,the rats from JLD group TC、TG、FFAreduced, and LDL-C increased(P<0.05) Jinlida could obviously improve the liver fat accumulation of mice. Compared with model group, Resistin、IL-6、TNFα、p-AMPKα、p-ACC、p-PI3K、p-Akt protein was significantly decreased. Compared with model group, p-AMPKα、p-AC、p-PI3K、p-Akt protein was increased in the mice liver from JLD group(P< 0.05, P< 0.01).3. The result of intervention study of Jinlida in the Palmitic acidinduced the injury in islet β cells:According to the results of CCK8,the cell survival activity of JLD groups are no difference with normal group; After injury of PA, INS-1 cell survival activity decreased, BAX, Atg7, Beclin-1 gene expression increased, Bcl-2 gene expression reduced(P<0.01), After medication, cell survival, Bcl-2,Atg7,Beclin-1 gene expression increased,and BAX gene decreased CompoundC could completelyabolishthestimulatingactivityof JLD on p62 gene acumulation. (P<0.01).Conclusion:1. This study is based on the modern famous doctor Zhang Xichun’s academic thought about diabetes that is "commence in middle jiao, and develop to the upper and lower", and based on the system studies of the etiology, pathogenesis and treatment research foundation of T2DM, and point that the key of disease onset is due to subtle distribution and metabolic disorder and imbalancecaused by dysfunction of spleen in transportation, put forward " subtle distribution and metabolic disorder and imbalance caused by dysfunction of spleen" is the main pathogenesis of T2DM, "phlegm and blood stasis block blood vessels caused by dysfunction of spleen " is the root cause of T2DM complications, and establish "activating spleen essence" therapeutic principle and synthesize some methods to cure"spleen",that is:raise spleen Yin, dry spleen dampness, drain spleen heat, warm the spleen Yang, form JLD particle, and analysis of JLD particles formula characteristics and clinical and experimental study progress, so as to provide effective treatment for T2DM.2.Experimental study:experiments in vivo show that JLD granuleeffectively adjust the T2DM rats blood glucose and insulin secretion, protect the beta cell structure, maintaining islet function, reduce islet cell apoptosis and protect the islet cells. JLD could adjust insulin resistance rats glucolipid metabolism, regulateadipokines, reduce liver lipid deposition, improve insulin sensitivity, its mechanism may be related to regulating AMPK/ACC and PI3K/Akt signaling pathway.Experiments in vitro:JLD increase the islet cell autophagy level, the efficacy of JLD is dependent on the activation of cellular AMPK pathway.Provides evidence for "diabetes commence in middle jiao", provides the experimental basis for the spleen for "treat T2DM from spleen", test the effects of the Jinlida for T2DM, and provides effective treatment for clinical treatment.