| DM is a multi-factorial disease characterized by hyperglycemia resulting fromdefects of insulin secretion and insulin action, which leads to impaired functions incarbohydrate, lipid and protein metabolism. It is estimated that the number ofdiabetics will rise to300million worldwide by2030, and95percent of those peoplebelong to T2DM. T2DM is characterized by insulin resistance, which is a state whereinsulin has a reduced ability to mediate glucose homeostasis in its major target tissues,such as skeletal muscle, adipose tissue and liver. Insulin resistance is not only the keypathophysiological abnormality of T2DM, but also the primary cause of many relatedcomplications, such as hyperglycemia, dyslipidemia, abdominal obesity, hypertension,and so on. Therefore, improving insulin resistance has become an established andacknowledgeable treatment for T2DM. The present therapies for DM include the useof insulin and oral hypoglycemia agents, but these approaches which currently used inclinical practice can also induce certain negative effects, such as hypoglycemia causedby higher doses of the drugs used in the therapies, liver problems, lactic acidosis anddiarrhea. Therefore, it is vital to seek for natural and non-toxic anti-diabetic medicinesfor diabetic therapy.Coptis Chinensis, also known as huanglian in chinease, one of the most widelyused traditional Chinese medicines, has attracted much attention because of itsmultiple pharmacological effects, such as anti-diabetic, anti-inflammation, anti-cancer,and so on. Previous studies have confirmed that various alkaloids, including berberine,coptisine, palmatine and jatrorrhizine could have beneficial physiological effects onDM and its complications. It is reported recently that Coptis Chinensis decoction canproduce much more obivious hypoglycemic activities than berberine, jatrorrhizine andtheir combinations. It is also demonstrated that its non-alkaloid extracts maysignificantly potentiate the ability of the cultivated3T3-L1adipocytes to uptakeglucose. The studies described above imply that there would be other constituents inCoptis Chinensis,which may contribute somehow to the treatment of DM and itscomplications, and the polysaccharide is dose one of the water-soluble components. There are few studies on biological activities of the polysaccharide extracted fromCoptis Chinensis, such as antibiosis, antioxidation and inhibition of cell proliferation,but no study on its anti-diabetic activity in DM.Therefore, in order to explore the antidiabetic effect of Coptis Chinensispolysaccharide and its mechanism, the establishment of extraction and separation ofCoptis Chinensis polysaccharide, its therapeutic effect on rats with T2DM, and itseffects on the insulin signaling pathway were evaluated in this study.1. The study of extraction and purification technology of Coptis ChinensispolysaccharideThe extractive and purified technology and the qualitative and quantitativeanalysis method of CCPW were studied in this part. The experimental results showedthat the best extraction technology should be the medical material crashed to40mesh,the water taken as the extraction solvent, the application of refluxing method, the solidto liquid ratio of1:10, the extraction temperature at100℃, extraction time for40minutes and3times of repeated extraction. The results of repetitive experimentconfirmed that the technology was stable and reliable, and the average yield of CCPWwas1.52%. The precipitation with ethanol, decoloration and deproteinization shouldbe performed after the extraction of CCPW to refine it. The experimental resultsshowed that the best purification technology included:3times of precipitation with95%ethanol, and decoloration with4%H2O2at45℃and in the solution with pH7.0for120min, and deproteinization by Sevag method and the ratio of deproteinizationwas81.3%. The quantitative and quality analysis verified that the Coptis Chinensispolysaccharide obtained with the above methods was a non reducibilitypolysaccharide, in which the content of total sugar was88.73%, the content of uronicacid was29.92%, and the content of protein was2.85%.2. The anti-diabetic effects of Coptis Chinensis polysaccharide on rats of T2DMThe combined high-fat diet (HFD) and streptozotocin (STZ)-induced methodwas used to establish a rats type2diabetes mellitus model successfully. After theCoptis Chinensis polysaccharide was administered to the diabetic rats, it was foundthat the mental conditions of the rats were improved markedly, the body weight losstrend of them was weakened, and the hyperglycemic levels of them were reduced bythe various doses of Coptis Chinensis polysaccharide; the glucose tolerance test(IPGTT) demonstrated that Coptis Chinensis polysaccharide could enhance the rats’abilities in glucose tolerance; moreover, Coptis Chinensis polysaccharide could the serum insulin levels and improve the insulin sensitivity index. Coptis Chinensispolysaccharide could reduce levels of NEFA/FFA, total glycerin (TG), and totalcholesterol (TC), suggesting that it should have a regulatory effect on the metabolismdisorders of blood lipids in the diabetic rats. After the administration of CoptisChinensis polysaccharide for4weeks, GSH contents and GSH-Px activities in theliver tissues of the rats were elevated; besides, super oxygen dehydrogenises (SOD)and catalase (CAT) activities were significantly enhanced, and methane dicarboxylicaldehyde (MDA) contents were significantly reduced. It was demonstrated that CoptisChinensis polysaccharide could improve the antioxidant enzyme activity and lowerthe lipid peroxide content, thereby strengthening the ability to scavenge free radicalsand reducing the production of ROS to play a role in the anti-oxidative stress and theprevention of oxidative damage, which might be the reason for its anti-diabetic effectsand improvement of insulin resistance.3. The effects of Coptis Chinensis polysaccharide on the insulin signaltransduction pathwayIn order to further explore the molecular mechanism of CCPW on anti-diabeticeffect and improving insulin resistance, effects of CCPW on JNK and Glut4expressions at the mRNA levels were measured with RT-PCR, and JNK,Phospho-IRS1(Ser307), IRS1, PI3Kp85, PI3K and Glut4expressions at the proteinlevel were detected with Western blot. The results showed that compared with those inthe model group, CCPW could inhibit the JNK expression and improve the Glut4atthe mRNA level, inhibit the Phospho-IRS1(Ser307) expression and improve theexpression of PI3Kp85and Glut4at the protein level, but not affect the expression ofIRS1and PI3K obviously. Many studies have demonstrated that oxidative stress isclosely related to diabetes and insulin resistance. The oxidative stress can increase theproduction of JNK, thus make IRS1phosphorylated, and finally result in the insulinresistance. It is further verified in our experiments that CCPW could reduce theexpression of JNK through its antioxidative stress and then inhibit thephosphorylation of IRS1(Ser307), leading to the increase of PI3Kp85expression, thenaffect the whole pathway of PI3K to increase the expression of Glut4, and finallyproduce the antidiabetic effect and improve the insulin resistance.In conclusion, the extractive and purified technology and the qualitative andquantitative analysis method for CCPW were established. The administration ofCCPW to the rats with T2Dm has demonstrated that it has an anti-diabetic effect and an effect on improving insulin resistance. The anti-diabetic effect and the effect onimproving insulin resistance of CCPW may be related to its antioxidative stress.CCPW can suppress the expression of JNK, reduce the phosphorylation ofIRS1(Ser307), thereby promoting the combination of InsR and IRS1, leading to theactivation of the PI3K pathway to increase the expression of Glut4, and finallyproduce the anti-diabetic effect and the effect on improving the insulin resistance. |
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