| Objevtive:To investigate the mechanism of X-ray-induced myocardial fibrosis and clarify the possible underlying mechanisms of Radix Angelica Sinensis and Radix Hedysari ultrafiltration extract(RAS-RH)on X-irradiation-induced cardiac fibrosis in rats.Method:1 Establishment of a cell model of X-irradiation-induced myocardial fibrosis:the heart tissue from neonatal rat was digested with trypsin,the cardiac fibroblasts were isolated by digestion method,X-ray-induced fibroblasts model in heart was establishd using a single total body irradiation of 0.5Gy,1Gy and 2Gy X-ray dose,the expression of TGF-β1 and Col1α in neonatal rat cardiac fibroblasts was analyzed by Western Blot,the content of TGF-β1 in the supernatant was also detected by ELISA.2 Inhibiting TGF-β1 and examining the effect on OPN,Col1α and miRNA-21-5p in neonatal rat myocardial fibroblasts: cardiac fibroblasts from neonatal rat was cultured,the expression of TGF-β1 in cardiac fibroblasts was inhibited using 1 μg,5 μg and 10 μg Disitrtide,the expression of OPN and Col1α in neonatal rat cardiac fibroblasts was detected by Western Blot,the expression of miRNA-21-5p in neonatal rat cardiac fibroblasts was detected by qPCR.3 Interfering OPN and detecting the effect on TGF-β1,Col1α and miRNA-21-5p: cardiac fibroblasts from neonatal rat was cultured,Si-NC or Si-r-Spp1 was transfected with Lipofectamine3000.The expression of TGF-β1 and Col1α in neonatal rat cardiac fibroblasts was detected by Western Blot.MiRNA-21-5p was measured by qPCR.4 Inhibiting miRNA-21-5p and detecting the effects on OPN,Col1α and TGF-β1 in neonatal rat myocardial fibroblasts: cardiac fibroblasts from neonatal rat was cultured,micrOFFTM mo-miRNA-21-5p was transfected with Lipofectamine3000.The expression of TGF-β1,OPN and Col1α in neonatal rat cardiac fibroblasts was detected by Western Blot.5 Inhibiting miRNA-21-5p and detecting the effect on TNF-α,Caspase-3 and ROS: cardiac fibroblasts from neonatal rat was cultured,inhibiting miRNA-21-5p in neonatal rat cardiac fibroblasts,the expression levels of TNF-α and Caspase-3 in neonatal rat cardiac fibroblasts was detected by Western Blot.The content of ROS was measured by Elisa.6 Establishing a Wistar rat model of radiation-induced myocardial fibrosis using a single total body irradiation of 6Gy,8Gy and 10 Gy X-ray dose,H&E and Masson staining identify the degree of myocardial fibrosis in rat heart tissue,and screen the optimal X-ray dose for a Wistar rat model.The rats were given RAS-RH(25 or 50 or 100 mg/kg/day)for 30 days.Morphological change and ELISA were used to screen concentration of RAS-RH,and serum TnT and BNP levels was analysed to identify X-ray-induced myocardial fibrosis in rats.Immunofluorescence staining was used to detect the expression of TGF-β1 and Col1α.7 To detect whether RAS-RH protects X-irradiation-induced myocardial fibrosis by regulating the TGF-β/OPN/AP-1/miRNA-21 pathway: rats were randomly divided into control group,X-ray group and RAS-RH+X-ray group,cardiac fibroblasts were digested by double enzyme and isolated from rats.CCK-8 was used to detect cardiac fibroblasts proliferation,the gene expression levels of TGF-β1,Spp1,c-jun,c-fos,miRNA-21-3p,miRNA-21-5p and Col1α in rat cardiac fibroblasts was measured by qPCR,the protein expression levels of TGF-β1,Spp1,P-c-jun,P-c-fos and Col1α in rat cardiac fibroblasts was tested by Western Blot.8 To detect whether RAS-RH alleviate X-irradiation-induced myocardial fibrosis by reducing oxidative stress damage: rats were randomly divided into control group,X-ray group and RAS-RH+X-ray group.The content of ROS in rat serum and SOD in rat cardiac fibroblasts was detected by ELISA.9 To detect whether RAS-RH inhibits the occurrence of X-irradiation-induced myocardial fibrosis by improving the apoptosis resistance of rat myocardial fibroblasts: rats were randomly divided into control group,X-ray group and RAS-RH+X-ray group.H&E staining was used to observe the apoptosis of myocardial fibroblasts,flow cytometry was also used to detect the apoptosis of myocardial fibroblasts.The expression of TNF-α and Caspase-3 in rat cardiac fibroblasts was measured by qPCR and Western Blot.Result:1 1Gy X-ray dose were used to establish a cell model of X-irradiation-induced myocardial fibrosis: compared with the control group,the levels of TGF-β1 and Col1α was not significantly change in the 0.5Gy X-ray group and significantly increased in the 1Gy X-ray group,the content of TGF-β1 was significantly increased and the expression of Col1α was not significantly change in the 2Gy X-ray group.X-ray-induced myocardial fibroblast model in neonatal rats with 1GyX-ray dose was established in the subsequent experiment.2 TGF-β1 is involved in the development of X-irradiation-induced myocardial fibrosis by regulating OPN and miRNA-21-5p: the expression of TGF-β1 of rat cardiac fibroblasts was inhibited by Disitrtide.Compared with the control group,the expression of OPN and Col1α in X-ray group was obviously increased.Compared with the Si-NC+X-ray group,the expression of OPN and Col1α from the cardiac fibroblasts in TGF-β1 inhibitor+X-ray group were significantly decreased.The miRNA-21-5p expression pattern was consistent with OPN.3 X-ray regulates the expression of TGF-β1 and miRNA-21-5p by OPN,which promotes the occurrence of X-irradiation-induced myocardial fibrosis.When the expression of OPN in neonatal rat cardiac fibroblasts was inhibited,compared with the Si-NC group,the expression of TGF-β1 and Col1α of the Si-NC + X-ray group was significantly increased with statistical significance.Compared with the Si-NC + X-ray group,the expressions of TGF-β1 and Col1α in the Si-Spp1 + X-ray group were reduced(P <0.05),and the expression pattern of miRNA-21-5p was consistent with TGF-β1.4 X-rays promote the expression of TGF-β1 through miRNA-21-5p,which mediate the occurrence of X-irradiation-induced myocardial fibrosis.When the expression of miRNA-21 of rat cardiac fibroblasts was inhibited,compared with Si-NC group,the expression of TGF-β1,OPN and Col1α from myocardial fibroblasts in the Si-NC+X-ray group were increased.Compared with Si-NC+X-ray group,the expression of TGF-β1,OPN and Col1α from cardiac fibroblasts in miRNA-21-5p inhibitor+X-ray group were reduced,and the expression of TGF-β1 and Col1α was statistically significance,the expression of OPN was not statistically significant.5 X-ray promotes the generation of ROS and reduces the expression of TNF-α and Caspase-3 in cardiac fibroblasts by miRNA-21-5p: compared with Si-NC group,the expression of TNF-α and Caspase-3 from neonatal rat cardiac fibroblasts in Si-NC+X-ray group were decreased.Compared with Si-NC+X-ray group,the expression of TNF-α and Caspase-3 in the miRNA-21-5p inhibitor+X-ray group were significantly elevated.On the other hand,inhibiting the expression of miRNA-21,compared with the Si-NC group,the ROS content of rat neonatal rat myocardial fibroblasts in the Si-NC + X-ray group was significantly increased,and it was statistically significant(P < 0.01).Compared with the Si-NC + X-ray group,the content of ROS in neonatal rat myocardial fibroblasts of miRNA-21-5p inhibitor + X-ray group were significantly reduced,which was statistically significant(P <0.01).6 8Gy X-ray was used to establish a rat model of X-irradiation-induced myocardial fibrosis: H&E and Masson staining showed that X-ray doses of 6Gy,8Gy and 10 Gy could induce myocardial fibrosis in Wistar rats.The degree of fibrosis in the 8Gy X-ray and 10 Gy X-ray groups was heavier than the 6Gy X-ray group,two rats in the 10 Gy X-ray group were died during X-ray induced acute inflammatory response,so we selected 8Gy X-ray doses in subsequent trials.7 50mg/kg of RAS-RH can effectively protect the occurrence of X-irradiation-induced myocardial fibrosis: H&E and Masson staining revealed that medium-high-dose of RAS-RH can relieve X-ray-induced myocardial fibrosis.ELISA results showed that: compared with the control group,X-ray can significantly increase the expression of TGF-β1.Compared with the X-ray group,50 mg/kg and 100 mg/kg of RAS-RH can significantly reduce the X-ray-induced TGF-β1 content,which was statistically significant(P <0.01),25 mg/kg no statistical significance.Compared with the 50mg/kg group,there was no statistically significant change in the content of TGF-β1 in the 100mg/kg group,so the 50 mg/kg dose of RAS-RH was used in the subsequent experiments.8 RAS-RH effectively protects against the occurrence of X-irradiation-induced myocardial fibrosis by reducing the expression of TGF-β1 and Col1α in rat myocardium and the level of TnT in rat serum: TnT and BNP levels in rat serum were measured to identify X-ray indued myocardial fibrosis rat models.Compared with the control group,the level of TnT was elevated in the X-ray group,which was statistically significant(P < 0.01).Compared with the X-ray group,the level of TnT was significantly lower in the RAS-RH+X-ray group(P < 0.05).H&E and Masson staining showed that the degree of cardiac fibrosis in the RAS-RH+X-ray group was reduced,RAS-RH is effective on protection of X-ray indued myocardial fibrosis in rats.9 RAS-RH inhibits the proliferation of rat myocardial fibroblasts: CCK-8 results showed that the proliferation activity of myocardial fibroblasts in X-ray group was significantly increased compared with the control group,and was decreased in the RAS-RH+X-ray group compared with the X-ray group.RAS-RH inhibits proliferation of cardiac fibroblasts.10 RAS-RH protects the occurrence of X-irradiation-induced myocardial fibrosis by regulating the TGF-β1/OPN/AP-1/miRNA-21 pathway: qPCR and Western Blot results showed that the expression of TGF-β1,OPN,P-c-jun,miRNA-21-3p,miRNA-21-5p and Col1α from cardiac fibroblasts in the X-ray group were significantly increased compared with the control group.Compared with X-ray group,the expression of TGF-β1,OPN,P-c-jun,miRNA-21-3p,miRNA-21-5p and Col1α in rat cardiac fibroblasts were remarkably decreased in the RAS-RH+X-ray group.There was no significant change for P-c-fos between X-ray group and RAS-RH+X-ray group;the expression of miRNA-21-5p and miRNA-21-3p was significantly higher in X-ray group when compared to the control group,after RAS-RH treatment,the expression of miRNA-21-5p and miRNA-21-3p was significantly lower in RAS-RH+X-ray group when compared to the X-ray group.11 RAS-RH relieves X-irradiation-induced myocardial fibrosis by reducing oxidative stress damage: compared with the control group,serum ROS contents in the X-ray group were significantly increased and the SOD content of cardiac fibroblasts was significantly decreased,with statistical significance(P < 0.01).Compared with the X-ray group,the serum ROS levels in the RAS-RH+X-ray group were decreased and the SOD content of cardiac fibroblasts in the RAS-RH+X-ray was increased,with statistical significance(P < 0.01).12 RAS-RH inhibits the occurrence of X-irradiation-induced myocardial fibrosis by improving the apoptosis resistance of rat myocardial fibroblasts:(1)H&E staining showed morphological change of myocardial fibroblasts.The control group showed normal change,the cell membrane is complete,the cytoplasm is light red,the nuclear chromatin staining is uniform,the nuclear membrane is complete,pale blue or blue,and scattered apoptotic cells can be seen.Compared with the control group,the number of myocardial fibroblasts apoptosis in the X-ray group wse reduced,the cell volume was reduced,the cytoplasm was dense,the eosinophilic staining was enhanced,the nucleus was condensed and fragmented,and it was blue-black,apoptotic bodies were observed,decentralized single form exists.Compared with the X-ray group,the number of myocardial fibroblasts apoptosis increased in the RAS-RH+X-ray group;(2)Flow cytometry showed that the apoptosis of primary cardiac fibroblasts in X-ray group was decreased compared with the control group,after RAS-RH treatment,compared with the X-ray group,the apoptosis of primary cardiac fibroblasts in the RAS-RH+X-ray group was increased;(3)The expression of TNF-α and Caspase-3 from primary cardiac fibroblasts in the X-ray group were decreased compared with the control group,which was statistically significant.Compared with the X-ray group,the expression of TNF-α and Caspase-3 from primary cardiac fibroblasts in the RAS-RH+X-ray group were increased,which was statistically significant.Conclusion:1 Conclusion in cell experiment(1)The profibrotic molecules of miRNA-21,TGF-β1 and OPN are key molecules for radiation-induced myocardial fibrosis,there is positive relationship between TGF-β1 and OPN and miRNA-21,OPN promotes the expression of TGF-β1 and miRNA-21,and miRNA-21 up-regulates the expression of TGF-β1,TGF-β1 and OPN can up-regulate the expression of miRNA-21,which promotes the occurrence of radiation-induced myocardial fibrosis.(2)miRNA-21 mediates the apoptosis and oxidative stress injury of rat cardiac fibroblasts.2 Conclusion in animal experiment(1)Radix Angelica Sinensis and Radix Hedysari ultra-filtration protects radiation-induced myocardial fibrosis by TGF-β1/OPN/AP-1/miRNA-21 pathway.(2)Radix Angelica Sinensis and Radix Hedysari ultra-filtration reduced oxidative stress injury of myocardial fibroblasts in rats and alleviated the occurrence of X-ray-induced myocardial fibrosis.(3)Radix Angelica Sinensis and Radix Hedysari ultrafiltration extract improves apoptosis resistance of X-ray-induced cardiac fibroblasts in rats and inhibited the occurrence of X-ray-induced myocardial fibrosis. |
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