The Research On The Protective Effect Of Kang Fu Ling Against Cardiac Injury Induced By HPM Radiation

Objective: With the advent of industrialized information society, the living environments of human are more and more contaminated by electromagnetic radiation. Studies have shown thatcertain intensity micro wave radiation can cause multiple organ damage. The heart is one of the more sensitive organs, however effective medicines for prevention and treatment are lack. Traditional Chinese medicines used for the treatment of cardiovascular diseases have achieved good results, besides In the case of lack of medicine for prevention and treatment of injury caused by microwave, development of traditional Chinese medicines used for prevention and treatment of heart damage induced by microwave radiation is feasible and necessary. In this study, regulation of GSK-3β to m PTP has been taken as the entry point to explore the protective effect of Kangfuling and its active ingredient on the heart and the mechanism of action, in order to explore the protection of Kangfuling and its active ingredient against HPM radiation-induced heart injury, to determine the effective dose and drug targets, providing an important experimental basis for the development of medicine against microwave-induced injury.Materials and methods:(1) The study on the protective effect of Kangfuling on the HPM radiation induced cardiac injury and mechanism in rats: 120 male Wistar rats were given HPM radiation with the average power density of 30 m W/cm2 for 15 min, and have been with 0.75、1.5、3g/kg/d Kang Fu Ling and 1.5g/kg/d ANDuo Lin, receiving medicine by filling stomach once a day for 14 days consecutively. At the time points of 7d after the beginning of administration(7d after irradiation), 6h after the end of administration(14d after irradiation), and 7d after the end of administration(21d after irradiation), electrocardiogram, Myocardial enzyme and Ca2+ in serum, cardiac tissue structure, mitochondrial ultrastructure, mitochondrial membrane potential and m PTP, and the levels of VDAC protein and phosphorylation of GSK-3β protein were detected to determinethe protective effect of kangfu Ling on the HPM radiation-induced heart damage, and the most effective dose, and to explore the possible mechanism of protection process.(2) The study on the protective effects of Astragalosides, Total paeony glycosides and Total tanshinones on HPM Radiation induced myocardial cell injury: The H9c2Myocardial cells were given HPM radiation with the average power density of 30 m W/cm2 for 6 min, and were administered with 1, 3, 9 mg/l Astragalosides, Total paeony glycosides and Total tanshinones 48 h before irradiation. At the time points of 48 h before irradiation and 30 min after irradiation, drug cytotoxicity, the content of ROS in cells, activity of Myocardial enzyme in culture medium were detected to determine the protective effects of Astragalosides, Total paeony glycosides and Total tanshinones on HPM radiation-induced heart injury, to find the ingredients with the best efficacy and the optimal dose for cellular level.(3)The study on the protective effect of Astragalosides on the HPM radiation induced cardiac injury in rats: 120 male Wistar rats were given HPM radiation with the average power density of 30 m W/cm2 for 15 min, and have been with 40, 80, 160mg/kg/d Astragalosides by filling stomach once a day for 14 days consecutively. At the time points of 7d after the beginning of administration(7d after irradiation), 6h after the end of administration(14d after irradiation), by electrocardiogram, myocardial enzyme and Ca2+ in serum, Cardiac histological structure and ultrastructure were observed to determine the protective effect of Astragalosides on the HPM radiation-induced heart damage, and the most effective dose.(4) The study on the Effect of the regulation of GSK-3β to m PTP in the protection of Astragalosides against HPM radiation-induced myocardial cell injury in rats: The H9c2 Myocardial cells were given HPM radiation with the average power density of 30 m W/cm2 for 6min, and were administered with 3mg/l Astragalosides 48 h before irradiation.24 h before irradiation, the GSK-3β specific inhibitor 1-Azakenpaullone with a concentration of 4μmol/l was used for intervention. At the time points of 30 min and 3h after irradiation, the expression of related proteins in GSK-3β regulating m PTP, m PTP open and its effect on mitochondrial function were detected. The aim was to explore the possible mechanism of the protective effect of astragalosides rat on HPM radiation induced cardiac injury in rats and to investigate the effective target in the protection of Astragalosides against HPM radiation induced heart damage.Results:(1) Kangfuling improves heart dysfunction and structural damage induced by HPM radiation in rats: serum CK-MB, LDH, AST activity and Ca2+ content were significantly increased 7d after 30 m W/cm2 HPM radiation( p<0.05 or p<0.01). 14 d after radiation, CK-MB and Ca2+ content were still higher than that of the normal control group(p<0.05); heart rate and J point displacement were significantly increased both at 7d and 14 dafter radiation(p<0.05 or p<0.01), and significantly improved both in Anduolin group and in the middle and high dose Kangfuling groups(p<0.05 or p<0.01); 7d after radiation, heart tissues in rats were observed under light micro scopy and electron micro scopy, myocardial cell edema, muscle fibers in a wavy arrangment, stripes blurred, some nuclear condensed, stained, peripheral gap widened, mitochondrial swelling, morphological abnormalities, such as racket-shaped, some mitochondria off the ridge, cavitation, vascular endothelial cell cytoplasmic vesicle increased, perivascular space widened, intercalated disk blurred were found. Those abnormal changes mentioned above were significantly improved both in Anduolin group and in the middle and high dose Kangfuling groups, cylindrical myocardial cells, no edema, muscle fibers arranged in order, clear stripes, oval nuclei, located in the cellcenter. Normal mitochondrial morphology, myofibrils arranged regularly, vesicle endocytosis in the vascular endothelial cells significantly reduced, no abnormal perivascular space, clear intercalated disk structure were observed.(2) Kangfuling impoved the m PTP open, abnormal membrane potential, abnormal levels of GSK-3β phosphorylation, and VDAC protein expression in the heart tissue in rats, which were caused by HPM radiation: 7d after 30 m W/cm2 HPM radiation, membrane potential was decreased, m PTP open was increased, the levels of p-GSK-3β and VDAC protein were significantly lower(p<0.05). Compared with the radiation control group, in Anduolin group and in the middle and high dose Kangfuling groups, membrane potential was increased, m PTP open was lowered, and the levels of p-GSK-3β and protein VDAC expression were significantly increased(p<0.05)(3) astragalosides, TPG glycosides and total tanshinones improved the abnormalities caused by HPM radiation of ROS content in myocardial cells and myocardial enzyme activity in the culture medium: 30 min after 30 m W/cm2 HPM radiation, cardio myocytes ROS content and CK and LDH activity in culture medium were significantly increased(p<0.05 or p<0.01); Astragalosides, Total paeony glycosides and Total tanshinones exhibit varying degrees of improvement. Compared with the radiation control group, ROS content in myocardial cells, in three dose Astragalosides groups, low dose Total paeony glycosides group, low and high dose total tanshinones groups were significantly lower(p<0.05 or p<0.01); myocardial enzyme activity was significantly lower in middle and high dose Astragalosides groups and in middle dose total tanshinonesgroup(p<0.05).(4) Astragalosides improved heart dysfunction and structural damage caused by HPM radiation in rats: 7d after radiation,activities of serum LDH, AST were significantly increased(p<0.05 or p<0.01). 14 d after radiation, Ca2+ content was significantly increased(p<0.01), AST activity was still higher than the normal control group(p<0.05); 7d after radiation, heart rate and P-wave amplitude were significantly increased(p<0.05 or p<0.01). 14 d after radiation, T wave amplitude was significantly increased(p<0.05), P-wave amplitude is still higher than the normal control group. In middle and high dose Astragalosides groups, the improving effect was significant(p<0.05 or p<0.01); 7d after radiation, heart tissue in rats were observed under light and electron microscopy, myocardial cell edema, muscle fiber in wavy arrangement, stripes blurred, some nuclear condensed, stained, mitochondria swelling or with abnormal morphology, vascular endothelial cell cytoplasmic vesicle increased, perivascular space widened, intercalated disk blurred were observed. in middle and high dose Astragalosides groups, the abnormal changes were significantly improved, cylindrical myocardial cells, no edema, muscle fibers arranged in order, clear stripes, oval nuclei, located in the cellcenter. Normal mitochondrial morphology, myofibrils arranged regularly, vesicle endocytosis in the vascular endothelial cells significantly reduced, no abnormal perivascular space, clear intercalated disk structure were observed.(5) Astragalosides improved the abnormalities caused by HPM radiation of myocardial viability, m PTP open, the membrane potential, levels of cardio myocytes GSK-3β phosphorylation, and VDAC protein expression, and mitochondrial dysfunction: 30 min after 30 m W/cm2 HPM radiation, cellular viability was reduced, m PTP open was observed, membrane potential was decreased, the level of GSK-3β phosphorylation expression in cardiac cells was reduced, ROS content in cells was elevated, ATP content was decreased and mitochondrial Ca2+ content was decreased. 3h after radiation, cellular viability was reduced, the levels of GSK-3β phosphorylation and VDAC protein expression in cardio myocytes were decreased, ROS content in cells was elevated, ATP content was decreased and mitochondrial Ca2+ levels were increased(p<0.05 or p<0.01). In 1-Azakenpaullone inhibitor groups and Astragalosides groups, these changes were statistically significantly inhibited(p<0.05 or p<0.01).Conclusion:(1) 30 m W/cm2 HPM radiation can cause heart dysfunction, structural damage in rat, opening of m PTP, reduced membrane potential, abnormal expression of p-GSK-3β and VDAC protein.(2) Kangfuling has a good protective effecton the HPM radiation-induced heart injury, which is similar to that of Anduolin, and the optimal effective dose of 1.5g/kg/d; protective mechanisms may be related to inhibition of m PTP opening caused by the inhibition of GSK-3β activity and promotion of VDAC protein expression.(3) 30 m W/cm2 HPM radiation can cause increased activities of CK, LDH in H9c2 cell culture medium and elevated levels of ROS in cells. After pretreatment using Astragalosides, TPG glycosides and Total tanshinones, improving effect on these changes is good, and the effect of Astragalosides is the best, the optimal effective dose is 3mg/l.(4) Astragalosides have good protective effect on heart dysfunction and structural damage induced by 30 m W/cm2 HPM radiation in rats, the optimal effective dose is 80mg/kg/d.(5) 30 m W/cm2 HPM radiation can cause a decrease of H9c2 myocardial cell viability, abnormalities of Intracellular ROS and ATP content and mitochondrial Ca2+ concentration,a decrease of mitochondrial membrane potential, m PTP open, significant reduction of levels of GSK-3β phosphorylation and VDAC protein expression, and Astragalosides has a good protective effect.(6) GSK-3β is the important target molecule in HPM radiation induced myocardial cell damage. GSK-3βhas participated in the process of Astragalosides improving HPM radiation induced myocardial injury, possibly by suppressing the activity through phosphorylation of GSK-3β, promoting VDAC protein expression, thereby inhibiting m PTP open and improving mitochondrial function.