| Purpose:This subject consider that "toxin injury heart collaterals" is the main pathogenesis of diabetic cardiomyopathy,therefore,the experimental application of Jiedutongluo drink,through the overall and micro two methods in diabetic cardiomyopathy rats as the research object,to explore the regulation mechanism of Jiedutongluo drink on signal pathway of PPAR ?-NF-? B diabetic cardiomyopathy rats and inflammatory factor the verification of "toxin injury heart collaterals" is the key pathogenesis of diabetic cardiomyopathy theory,enriches the research ideas of diabetic cardiomyopathy and to provide experimental basis for clinical treatment of diabetic cardiomyopathy.Method:1.Experimental Study on the mechanism of Jiedu Tongluo Decoction on experimental diabetic cardiomyopathy in rats100 male Wistar rats(weight 200g ± 20g)were selected at the age of 8 weeks,and were randomly divided into normal control group(n =8)and model group(n = 92).A diabetic rat model was established by intraperitoneal injection of streptozotocin(STZ)55mg · kg-1,Each group was fed for 1 weeks,The detection of random blood glucose,two random blood glucose 16.7mmol/L,and accompanied by polyuria,polydipsia,multi feeding symptoms of rats is a successful model of diabetic rats,the establishment of a total of 77.The rats were fed with normal diet for 12 weeks to induce diabetic cardiomyopathy(DCM)model,and the RBG and body weight(body,BW,weight)of rats in each group were monitored every week,and a total of 51 DCM rats were divided into two groups by the end of 12 weeks.The DCM model rats were randomly divided into:Jiedutongluo drink group,Qingrejiedu drug group,pioglitazone group,DCM model group,were orally given Jiedutongluo drink 10.4g/(kg · d),2.7g/(kg · d),pioglitazone 2.7mg/(kg · d),DCM group and NC group were given equal volume of distilled water,the time of drug intervention for 6 weeks.At the end of the eighteenth week,the rats were observed and recorded.The left ventricular pressure was measured by left ventricular catheterization;measured the heart weight of rats;Detection of serum glycosylated serum protein(Glycosylated Serum,Protein,GSP),blood lipid and high sensitive C reactive protein(hs-CRP),interleukin-1 beta(IL-1 beta)and tumor necrosis factor alpha(TNF-alpha);equatorial plane for paraffin pathological sections were cut by hematoxylin and eosin(HE),Masson staining,myocardial glycogen staining(PAS staining)and transmission electron microscopy and immunohistochemistry to detect the expression of PPAR and NF-gamma kappa B expression in myocardial tissue mRNA extraction;fluorescence quantitative PCR detection of PPAR gamma,TNF-alpha,IL-1 beta and IL-6 gene;expression of myocardial tissue protein extraction for Western blot detection of PPAR gamma,NF-kappa B protein.2.The effect of Jiedutongluo Drink on pathway of PPAR ?-NF-? B-inflammatory factor in high glucose stimulated H9c2 cellsWith different concentrations of glucose(5.5,10,20,30,50mmol/L)stimulation of H9c2 myocardial cells 24 hours and 48 hours,using the method of MTT activity detection of different glucose concentrations on H9c2 cells;pharmacological evaluation of serum was detected by MTT method;according to the above results,the concentration of glucose was 30mmol/L,serum concentration cultured myocardial cells of H9c2 medium was 10%,select the 3-4 generation H9c2 myocardial cells,glucose concentration was 30mmol/L,containing 10%fetal bovine serum high glucose medium cultured for 24 hours,24 hours after the drug intervention group was given glucose concentration was 30mmol/L group serum concentration for 10%of the medium in cultured myocardial H9c2 after 24 hours,containing 10%fetal bovine serum DMEM low sugar culture medium as normal control group;the detection of IL-1 in the cell culture medium and TNF-alpha beta by Elisa method with The expression of TNF-alpha,IL-1 beta and IL-6 mRNA in myocardial cells was detected by RT-PCR method.The expression of PPAR,NF-kappa B protein in myocardial cells was detected by Western blot.Result:1.Experimental Study on the mechanism of Jiedu Tongluo Decoction on experimental diabetic cardiomyopathy in rats?4th weeks,8th weeks and 12th weeks,there was a significant difference between the normal control group and the model group in fasting weight,random blood glucose,drinking water,food intake and urinary output(P<0.01),the model group random blood glucose continued to increased,weight decreased weekly;after administration,13th weeks,16th weeks,18th weeks,compared with the normal control group,DCM model group significantly decreased body weight,random blood glucose increased significantly(P<0.01);the control group compared with the DCM model,after administration of Jiedutongluo drink Group,Qingrejiedu drug group and pioglitazone group random blood glucose were decreased significantly(P<0.01),no significant difference in fasting body weight(P>0.05).? The end of eighteen weeks.Quantity of drinking and eating,urine output results:compared with normal control group,DCM model group water intake,food intake,urine volume increased significantly(P<0.01);compared with the DCM model control group,Jiedutongluo Drink group,Qingrejiedu drug group and pioglitazone group of water intake,food intake,urine volume decreased significantly(P<0.01,P<0.05);heart function and cardiac index(H/W)test results:compared with normal control group,DCM model control group left ventricular function was significantly decreased,LVEDP and t-dp/dt were significantly increased,suggesting that the left ventricular diastolic dysfunction,LVSP,+dp/dtmax,-dp/dtmax decreased significantly left ventricular systolic function,H/W increased significantly,suggesting that the heart increased significantly(P<0.01,P<0.05);compared with the DCM model control group,Jiedutongluo drink group,Qingrejiedu drug,and pioglitazone group rats left ventricular diastolic dysfunction significantly improved,LVEDP and t-dp/dt were significantly lower(P<0.05),LVSP,+dp/dtmax,-dp/dtmax increased significantly,H/W decreased significantly(P<0.01,P<0.05);serum lipids,high sensitive C reactive protein(hs-CRP),glycosylated serum protein(GSP)the results of IL-1,TNF-and beta alpha:compared with the normal control group,DCM model group serum TG,TC,LDL-C,hs-CRP,GSP,IL-1 beta and TNF-levels increased significantly,HDL-C decreased significantly(P<0.01,P<0.05);compared with DCM model control group,Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group in serum TG,TC,LDL-C,hs-CRP,GSP,IL-1 beta and TNF-alpha,HDL-C were significantly increased(P<0.01,P<0.05);observation of left ventricular longitudinal slice HE staining under light microscope,the normal control group rats.Muscle cells arranged in neat,clear structure;DCM model control group of myocardial cells arranged disorder,myocardial hypertrophy,distortion,compared with the DCM model,Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group,myocardial cell injury has improved,myocardial cells were regular;Masson staining;the light microscope,compared with normal control group,DCM model control group cell hypertrophy deformation,interstitial fibers increased,interstitial fibrosis,Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group interstitial fibrous interstitial fibrosis has slightly thickened,improved;PAS staining,compared with the normal control group.DCM model control group glycogen volume fraction increased significantly(P<0.05);compared with the DCM model control group,Jiedutongluo drink group,Qingre jiedu drug group and pioglitazone group glycogen volume fraction decreased significantly(P<0.05);Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group glycogen volume fraction had no significant difference(P>0.05).?Immunohistochemistry results showed that:compared with the normal control group,DCM model control group of myocardial PPAR? expression was significantly decreased,NF-? B expression was significantly increased(P<0.05);compared with the DCM model control group,the expression of Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group were significantly increased myocardial PPAR ?,the expression of NF-? B was significantly decreased(P<0.05).?RT-PCR results showed that:compared with the normal control group,DCM model control group the expression of PPAR ? mRMA in myocardial tissue was significantly decreased,the expression of TNF-alpha and IL-1 beta and IL-6 mRNA increased significantly(P<0.05);compared with the DCM model control group,Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group the expression of PPAR Y mRMA in myocardial tissue significantly increased the expression of TNF-alpha,IL-1 beta and IL-6 mRNA decreased significantly(P<0.05).?Western blot results showed that:compared with the normal control group,DCM model control group DCM PPAR Y protein expression decreased significantly,the expression of NF-? B protein expression was significantly increased(P<0.01);compared with the DCM model control group,Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group,the expression of PPAR Y protein in myocardial tissue were significantly increased and the expression of NF-? B protein were significantly decreased(P<0.01).2.The effect of Jiedutongluo Drink on pathway of PPAR ?-NF-? B-inflammatory factor in high glucose stimulated H9c2 cells? The results of Elisa showed that:compared with the normal control group,model control group,cell culture medium IL-1 ? and TNF-a increased significantly(P<0.01,P<0.05);compared with the model control group,Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group cell culture medium IL-1 ? and TNF-? decreased significantly(P<0.01,P<0.05).?The RT-PCR results showed that:compared with the normal control group,model control group,the expression of IL-1 ? and TNF-? and IL-6 mRNA in myocardial cells were significantly increased(P<0.05);compared with the model control group,the expression of Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group the expression of IL-1 ? and TNF-? and IL-6 mRNA in myocardial cells decreased significantly(P<0.05).? The Western blot results showed that:compared with the normal control group,model control group,the expresson of PPAR Y was significantly decreased,NF-?B protein expression was significantly increased(P<0.05,P<0.01);compared with the model control group,the expression of Jiedutongluo drink group,Qingrejiedu drug group and pioglitazone group in myocardial cell PPAR Y protein were significantly increased,the expression of NF-? B protein was significantly decreased(P<0.01).Conclusion:1 Jiedutongluo drink can improve the general condition of diabetic cardiomyopathy in rats,and had the function of regulating glucose,lipid metabolism and anti-inflammatory;2 Jiedutongluo drink can reduce heart disease index in rats with experimental diabetic myocardium,improve cardiac function and inhibit cardiac hypertrophy,interstitial fibrosis and glycogen deposition,can protect the structure and function of myocardial function;3 Jiedutongluo drink can obviously increase the expression of PPAR? gene and protein in myocardial tissue of DCM rats,and inhibit the expression of NF-? B(p-p65)gene and protein and also inhibit the expression of IL-1 ? and TNF-? and IL-6 in myocardial tissue of DCM rats;4 Jiedutongluo drink can significantly increase the expression of PPAR Y protein in high glucose damaged H9c2 cells,and down regulate the expression of NF-? B(p-p65)protein,IL-1 ? and TNF-? and IL-6 in high glucose damaged H9c2 cells;5 The mechanism of Jiedutongluo drink on treating DCM may be related to the regulation of PPAR ?-NF-? B-inflammatory factor.6 "Toxin injury heart collaterals" is the key pathogenesis of diabetes disease,is closely related to the theory of inflammatory mechanisms in diabetic cardiomyopathy. |
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