| Part1:Literature researchAlthough the pathogenesis of type 2 diabetes mellitus (T2DM) is still the key difficulty to explore, glucolipotoxicity and insulin resistance (IR) from glyeolipid metabolic disorder of various metabolic diseases play important roles in the course of pathogenesis. Current studies suggested that the mechanisms of glyeolipid metabolic disorder were closely related with oxidative stress, inflammatory reaction and cell autophagy, and involved many cell signal transduction pathways, which had the interaction effect. AMP-activated protein kinase (AMPK) pathway, as a part of them, has attracted much attention due to its sensitivity to the cellular energy change and the function of modulating glucolipid metabolism signal pathways. With the research going on, there are many interventions including diet, exercise, drugs and natural extracts have been confirmed that they can improve glucolipid metabolic disorder of T2DM by regulating AMPK pathway. In combination with clinical manifestation and knowledge of contemporary doctor, T2DM glucolipid metabolic disorder belong to the diseases of TCM, such as Fei-pang, Pi-dan, Xiao-ke, Gao-zhuo, Xiao-dan and so on. The etiology of this disease due to improper diet, mental stimulation and lack of exercise, the TCM pathogenesis of this disease is caused by liver-spleen-kidney deficiency and phlegm turbidity and blood stasis. Combined with the understanding of glucolipid metabolic disorder from modern medicine, contemporary scholars put forward many new thoughts, based on the traditional theory of TCM, which have been proved effective. These are worthy of summarizing and studying. Among these characteristic theories and treatments, Jiangtangsanhuang Tablet (JTSHT) which is designed by Shanghan team from Guangzhou University of Chinese Medicine has developed a school of its own, and has solid scientific foundation and good clinical curative effect in the treatment of diabetes and its complications.Part2:Experiment researchObjective:The experiment used T2DM rats model, which treated by JTSHT. Observing the degree of glycolipid metabolism, insulin resistance, liver tissue pathological changes and protein/gene expression of hepatic AMPK, PGC-1 a in the model rats, in order to investigate the mechanism of glyeolipid metabolism disorder in T2DM rats. This can provide theoretical support of modern biology for the clinical applications of JTSHT.Methods:70 SPF SD male rats were fed adaptively for 7 days. Selected 10 rats which were fed with normal diet, as blank control group using simple randomization. The other 60 rats were fed with high-sugar and high-lipid diet for a month. The rats which were processed by high-sugar and high-lipid diet were intraperitoneally injected STZ (35 mg/kg) in one go, control group were injected citric acid buffer meanwhile. If fasting blood-glucose ≥11.1 mmol/L after 72 h combined with other symptoms such as polydipsia, diuresis, and this condition of blood-glucose could maintain for 2 weeks, they could be considered as type 2 diabetes model. Used random number table, the rats were divided into model group, low-dose group of JTSHT, high-dose group of JTSHT and metformin group. High-dose group and low-dose group were given 1.563 g/kg and 0.781 g/kg per day, respectively; metformin group were given 0.156 g/kg·d; blank control group and model group were given drinking water. After 8 weeks treatment, there were control group (10), model group (8), low-dose group (9), high-dose group (8) and metformin group (9) rats finishing the experiment. During the experiment, body weight, drinking, food intake and fasting blood-glucose of each rats were monitored before treatment,4 weeks and 8 weeks after initiation of therapy.8 weeks after treatment, blood lipids (TC, TG, LDL-C and HDL-C) and serum insulin were detected. Liver histomorphology changes were observed with HE staining. AMPK and pAMPK were detected by Western blotting method. AMPK and PGC-1α were detected by real-time PCR method.Results:1. The improvement of the glucolipid metabolism function:after 8 weeks of treatment, the weight of high/low-dose group of JTSHT and metformin group was significantly higher than model group(P<0.01). After 4/8 weeks of treatment, the drinking of high/low-dose group of JTSHT and metformin group was significantly less than model group (P<0.01). After 4 weeks of treatment, the eating of high/low-dose group of JTSHT and metformin group was significantly less than model group (P<0.05), and after 8 weeks of treatment, the eating of group of JTSHT had no difference with model group, and the eating of metformin group was significantly more than model group (P< 0.05). After 4/8 weeks of treatment, FBG of high/low-dose group of JTSHT and metformin group was significantly lower than model group (P< 0.05). After the treatment, TC, TG and LDL-C levels of high/low-dose group of JTSHT were significantly lower than model group (P<0.01 or P< 0.05), meanwhile TG and LDL-C levels of metformin group were lower than model group (P< 0.01). After the treatment, FINS and HOMA-IR index of high/low-dose group of JTSHT and metformin group was lower than model group, with statistical significance (P< 0.01).2. The improvement of the morphological changes of liver tissue:analysing the hepatosomatic index, compared with model group, the hepatosomatic index of high-dose group of JTSHT and metformin group was significantly lower (P <0.01). Through observing the anatomical morphology of liver, compared with normal liver, the liver of model rats looked more hypertrophied, toughened and the color of them was lighter accompanied with spots; compared with model group, the liver of treatment groups had the sharp edges and looked smaller, redder, but their surfaces looked mottled if closely observed. Through observing with the HE staining, model group existed different degrees of hepatic steatosis, while the hepatic steatosis degree of high/low-dose group of JTSHT and metformin group became lighter.3. The improvement of the hepatic AMPK protein expression. The hepatic AMPK and activated pAMPK protein expression levels of high/low-dose group of JTSHT and metformin group were higher than model group, with statistical significance (P< 0.01).4. The improvement of the gene expression of AMPK/PGC-1α signal path. The hepatic AMPK mRNA expression level of high/low-dose group of JTSHT and metformin group was higher than model group(P＜0.01). The hepatic PGC-1 a mRNA level of high-dose group of JTSHT and metformin group was lower than model group, with statistical significance (P< 0.01), while the difference between low-dose group of JTSHT and model group wasn’t significant.Conclusion:1. Based on the circular motion theory, the evolution of pathogenesis followed this rule:in the early stage, the middle pivot ran slowly, causing endogenous stasis; in the middle stage, the middle pivot ran out of circularly, causing swelter; in the late stage, the middle pivot ran unsuccessfully, causing deficiency of both Yin and Yang. The curative effect of JTSHT contained the guidelines of dredging for transporting Yang-qi, saving body fluid for protecting Yang-qi and stuffing soil for enriching Yang-qi, which could be mainly used in the early and mid stage of the glucolipid metabolic disorder. Recognizing the connotation of JTSHT, combined with the previous clinical and experimental research at the same time, was the premise condition which could be used to explore the mechanism of JTSHT for improving T2DM glucolipid metabolic disorder.2. JTSHT could increase the expression of hepatic AMPK and improve the activities of AMPK at the same time. It could also inhibit the PGC-1α gene overexpression in liver, thereby JTSHT played an important role in reducing blood sugar, blood lipid, the level of insulin resistance, improving clinlcal symptoms and delaying the liver tissue pathological changes in T2DM rats. Its mechanisms might involve the process of down-regulating gluconeogenesis and up-regulating fatty acid oxidation which participated by AMPK-PGC-1α. Due to reasons such as sample size and dose distribution, this experiment was temporarily not sure that JTSHT had definite concentration-response relationship. |
