| Purpose: Diabetes Mellitus(DM)is a metabolic disease mainly marked by hyperglycemia,and cardiovascular complications are the primary cause of death in diabetic patients.Ganoderma lucidium spores(GLS),a kind of fungal germ cells,showed a variety of medicinal functions,such as hypoglycemic,anti-inflammatory,etc.However,the protective mechanism of GLS against myocardial injury in diabetes has not been fully elucidated.In this study,protective effects of GLS on myocardium of diabetic rats and mechanism of PTEN-induced putative kinase1(PINK1)/parkin signaling pathway were discussed through the establishment of type 2diabetic rat models.It provides theoretical and experimental support for the potential of GLS in the treatment of diabetic myocardial injury.Methods: Forty male SD rats were randomly divided into normal group(NC,n=10)which were fed with ordinary diet;high fat and high sugar diet group(HFD,n=10)which were fed with high fat and high sugar diet.The remaining 20 rats combined with high fat and high sugar diet and intraperitoneal injection of 30mg/kg streptozotocin(STZ)induced type 2 diabetes model.Administration after 3 and 7 days,the blood glucose was higher than 16.7mmol/L for two consecutive times,and the model was considered successful,and the rats were randomly divided into diabetes group(DM,n=10)and GLS intervention group(GLS,n=10),and both groups fed with high fat and high sugar diet.GLS group was given GLS(300mg/kg)by intragastric administration,and the other three groups were given normal saline(10ml/kg)by intragastric administration.After 12 weeks of intervention,their body weight and fasting blood glucose(FBG)were recorded.Blood samples were collected from inner canthus veins after anesthesia,and serum samples were collected by centrifugation to evaluate creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH)levels.The rats were killed,the heart tissue was collected,and the cardiac organ coefficient was calculated.Malondialdehyde(MDA)and Superoxide dismutase(SOD)contents in myocardial tissue of rats in each group were measured by the kit.Hematoxylin-Eosin(HE)and Masson staining were used to observe myocardial pathological changes in all groups.The expression levels of PINK1/parkin signaling pathway and microtubule-associated protein 1 light chain 3,beclin1,ATG5 and p62 autophagy marker factors in the left ventricular myocardial tissue of rats in each group were detected by Western Blot(WB)and Immunohistochemistry(IHC).The ratio of apoptosis factor Bax/Bcl-2was detected by WB,and the apoptosis of myocardial cells in each group was further observed by TUNEL fluorescence staining.Results:(1)FBG,body weight and cardiac organ coefficient: Compared with NC group,FBG in HFD group had no significant fluctuation(P>0.05),and body weight and cardiac organ coefficient in DM group were significantly increased(P<0.01),while FBG and cardiac organ coefficient in DM group were significantly increased,and body weight was significantly decreased(P<0.01).Compared with DM group,FBG and cardiac organ coefficient were significantly decreased in GLS group,and body weight was significantly increased(P<0.01).(2)Detection of myocardial injury indexes: compared with NC group,the levels of CK-MB and LDH in HFD group and DM group were significantly increased,and the changes in DM group were more significant(P<0.01).Compared with DM group,the levels of CK-MB and LDH in GLS group were significantly decreased(P<0.01).(3)Myocardial histopathological test:myocardial cells in NC group were closely arranged with complete structure,and only a few collagen fibers were distributed around blood vessels.The arrangement of HFD group was slightly disordered,with occasional muscle fiber breakage,a few red blood cells in the interstitium,and increased collagen content.The arrangement of DM group was significantly disordered,with large muscle fiber breakage and a large number of red blood cell infiltration,and the collagen content was higher than that in NC group and HFD group(P<0.01).Compared with the DM group,the morphology and arrangement of GLS group tended to be normal,and the collagen content was significantly decreased(P<0.01).(4)Detection of PINK1/parkin pathway by WB and IHC: Compared with NC group,the protein expressions of PINK1,parkin and p-parkin in HFD group and DM group were significantly increased,and the changes in DM group were more significant(P<0.01).Compared with DM group,the expressions of PINK1,parkin and p-parkin proteins in GLS group were decreased(P<0.01).(5)Autophagy levels detected by WB and IHC: compared with NC group,LC3Ⅱ/Ⅰ,beclin1 and ATG5 protein expressions in HFD group and DM group were significantly increased,while p62 expression levels were decreased,and the changes in DM group were more significant(P<0.01).Compared with DM group,the protein expressions of LC3Ⅱ/Ⅰ,beclin1 and ATG5 in GLS group were decreased,while the expression level of p62 was increased(P<0.01).(6)Oxidative stress detection: compared with NC group,MDA content in HFD group and DM group was significantly increased,while SOD activity was significantly decreased,and the change was more significant in DM group(P<0.05).Compared with DM group,MDA was significantly decreased and SOD was significantly increased in GLS group(P<0.01).(7)Compared with NC group,the Bax/Bcl-2 ratio and myocardial cell apoptosis rate in HFD group and DM group were significantly up-regulated,and the changes in DM group were more significant(P<0.01).Compared with DM group,Bax/Bcl-2 ratio and myocardial cell apoptosis rate in GLS group were significantly decreased(P<0.01).Conclusion:(1)GLS can reduce blood sugar,improve myocardial hypertrophy,relieve pathological injury of myocardial tissue and reduce fibrosis degree in diabetic rats,so as to protect diabetic heart.(2)GLS can improve excessive autophagy of cardiomyocytes,exert good antioxidant activity,and reduce apoptosis of cardiomyocytes,which may be achieved by inhibiting PINK1/parkin signaling pathway. |
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