| Objective This research took the spontaneous obese type 2 diabetic mice as the experimental subject and observe the protective effect of HPS and DCM.The study also screened out the gene concerning the DCM myocardial fibrosis in model animals with the help of the PCR Array genetic chip technique and search for the important genes and approaches of controlling the oxidative stress and the inflammatory response.The thesis verifies the PPARγ/NF-κB signaling pathways and expression in the cardiac muscle and the influences that result from HPS on relevant index.This thesis aims at discussing the mechanism of HPS intervenes the DCM myocardial fibrosis and demonstrating the rationality of the HPS,the effective constituent that can cure the diabetes in Radix Hedysari,treatment of Qi deficiency,blood stasis and cult poison.We revealing the scientific connotation of the pathogenesis of diabetes and its complications,Qi deficiency,blood stasis and cult poison,which provides a scientific basis of preventing and treating DCM by HPS.Methods This research have following three parts,1)60 male db/db mice were random divided into five groups for observing the protective effects of Radix Hedysari on myocardial fibrosis of type 2 diabetic mice(db/db mice):a HPS high dose group,a HPS middle dose group,a HPS low dose group,a rosiglitazone group and a model group.A normal group was set up for paralleling observing.After 8 weeks of lavage administration,rats are killed by cervical dislocation and then put out the heart to detect some indexes of the blood: the content of LDH,CK-MB,TC and TG was detected by automatic biochemical analyzer,Hyp detected by the method of ELISA,the pathological changes and the fibrosis stage of the myocardial tissue stained by HE or Masson methods,the ultrastructural changes of myocardial tissue observed by transmission electron microscopy.The expressions of Collagen I,Collagen III m RNA and protein were detected by q PCR and Western blot,then did the study of the expression of Collagen I and Collagen III and positive staining area of collagenous fiber stained by Masson method.2)6 rats were divided into two groups: a model group consisted of 3 db/db rats and a normal group consisted of 3 db/m rats.After 8 weeks of lavage administration,rats are killed by cervical dislocation,we put out the heart to screen the gene that express differently in db/db rats and db/m rats by the technique of PCR Array gene chip,then discuss key genes of pathogenesis of DCM myocardial fibrosis and search for the signal path that PPARγ can control oxidative stress and the inflammatory response.Those provide a precise target and a regulaton mechanism for further study of preventing and treating DCM by HPS.3)The treatment method is same as part 1).After 8 weeks of lavage administration,rats are killed by cervical dislocation,we put out the heart to detect the activity of SOD and GSH-PX,the content of MDA,TNF-α and IL-6,the expressions of PPARγ,NF-ΚB,GLUT-4,IKKβm RNA and proteins by Real Time-PCR and Western Bolt.We analyzed the correlation between all kinds of indexes and myocardial fibrosis while we also analyzed the correlation between PPARγ/ NF-ΚB and inflammatory response and oxidative stress.Result1)(1)The blood glucose of high,middle dose HPS and rosiglitazone group were significantly lower than model group in 2,4,6 and 8 weeks(P<0.05 or P<0.01).Compared with the normal group,the level of TG,TC,LDH and CK-MB of model group increased distinctively(P<0.01)and the counterparts of high dose,middle dose HPS and rosiglitazone group both decreased distinctively(P<0.01).Especially in lowering the serum TC,LDH and CKMB,the decrease of high dose group was the most outstanding(P<0.05 or P<0.01).(2)Compared with normal group,collagen fiber positive staining areas were distinctively more(P<0.01),and the content of Hyp in each intervention group decreased obviously(P<0.01)especially in HPS high dose group.(3)The histological results showed that the appearances of rats’ cardiac muscle cells which were stained by HE or Masson method in the model group were hypertrophy,structure blurring,unclear margins,nuclear boggy,even shattering,melting and some parts significantly had the swelling,deformity,muscle fiber breakage,separation and inflammatory cellular infiltration in intercellular substance under optical microscope.The rats’ intracellular substance which was stained by Masson method of model group had more light green collagen fiber than normal group(P<0.01).The content of Hyp of all intervening groups significantly lowered(P<0.01),especially in high dose group.(4)The observation under the electron microscope demonstrated that the cardiac fiber arrangement showed the scattering,the distortion, lowered apparently in the cardiac tissue in the model group(P<0.01).Compared with the model group,the expressions of PPAR-γand GLUT-4 MRNA improved a lot whereas the expressions of NF-κB,IKKβ-m RNA and relative proteins lowered a lot in all intervening groups(P<0.01)except HPS low dose group.Besides,the effect form HPS high dose group was superior to the rosiglitazone group(P<0.01).The relation between the Hyp content and the TNF-α,IL-6 and MDA content was positive correlation(P<0.01).(3)The content of Hyp in myocardium was positively related to the levels of TNF-,IL-6 and MDA(P < 0.01).The relation between the Hyp content and the SOD,GSHx was negative correlation in cardiac muscle(P<0.01).The collagen fiber positive staining areas in the cardiac tissue had the negative correlation with the PPAR-γ expression(P<0.01)which had a positive correlation with the levels of SOD and GSHx(P<0.01),negative correlation with the level of MDA(P<0.01)and the positive correlation with the NF-κB expression(P<0.01)which had a positive correlation with the level of IL-6 and TNF-α(P<0.01).Conclusion1.HPS can reduce myocardial cell and tissue injury of animal model and slow the deposition of myocardial collagen and the interstitial fibrosis down.One of the mechanisms is the inhibition of expressions of Collagen I and Collagen III.2.Through PCR Array chip which was designed in custom in this study screened,it was preliminarily found PPARγ/NF-κB and the downstream inflammatory response,oxidative stress,fatty acid metabolism and other phenotypic gene of the regulation from them in total 64 has involved in the process of myocardial fibrosis in diabetic cardiomyopathy DB mice.3.HPS can significantly reduce the inflammatory response of DCM in myocardial tissue and improve the oxidative stress state,which may be related to the regulation of PPARγ/NF-κB signaling pathway and downstream related cytokines.4.The expression of glucose and lipid,Collagen I,Collagen III,TNF-α,IL-6,MDA,NF-κB,IKKβ and MMP9 enhanced is pathological basis to speed up DCM myocardial cells and tissue injury,myocardial tissue inflammation,oxidative stress and myocardial collagen deposition and fibrosis of the animal model,which is the molecular basis of diabetes and chronic complications of “blood stasis and toxin ”.While PPARγ,GLUT-4,SOD and GSH-PX are the molecular basis of diabetes and chronic complications of “virtual”,the effect of invigorating Qi and removing blood stasis of hedysari’s active ingredient of HPS can play a certain regulatory role in the expression of these factors which was not balanced in DCM myocardium of mice tissue that can take its expression of equilibrium,which may be the effective mechanism of the inhibition process of myocardial fibrosis. |
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