| Objective: The purpose of this study is to establish an animal model of exhaustive exercise training in mice,to explain the protective effect of CBD on myocardial injury in mice,and to explore its possible molecular mechanism,so as to provide experimental basis for the medicinal treatment of exercise-induced myocardial injury with CBD.Methods: Twenty-four healthy male C57BL/6 mice aged 6-8 weeks were randomly divided into control group(group C,n = 8),exhaustive exercise training group(RE group,n = 8)and exhaustive exercise training CBD group(REC group,n = 8).The animal model of exhaustive exercise training in mice was established by using animal treadmill.The mice were trained adaptively for 7 days(10m pinch minary 30mpmpd).The formal training was done with a slope of 10 degrees,and the speed of 10m/min was 10 min and15mpmin.After 10 min,the mice were finally exhausted at the speed of 20m/min.30 minutes before exercise,the REC group received intraperitoneal injection of 50mg/kg cannabinol solution(solvent 1% Tween 80 mg 20% ethanol + normal saline).The REC group received intraperitoneal injection of 50mg/kg cannabinol solution(solvent 1%Tween 80% ethanol + normal saline).The concentration of the drug is 12.5mg/ml.The RE group was intraperitoneally injected with the same amount of placebo(1% Tween 80%ethanol + normal saline)and was given twice a week.The above program exercises 6 days a week,and the training ends after 3 weeks.24 hours after exhaustive exercise training,the heart function of mice was evaluated by echocardiography.The hearts of mice were killed and separated.The pathology and collagen deposition of myocardium were evaluated by HE staining and Masson staining,and the changes of cross-sectional area of myocardial tissue were evaluated by WGA staining.Myocardial interleukin-6(IL-6),interleukin-10(IL-10),tumor necrosis factor-?(TNF-?),Bax,caspase-3(caspase-3),Bcl-2,malondialdehyde-5(MDA-5),endoplasmic reticulum stress-related factor(IRE-1-?),NADPH oxidase 2(NOX-2),superoxide dismutase(SOD-1),Kelch-like epichlorohydrin-related protein 1(Keap1)were detected by Westernblot.Protein expression of intranuclear protein nuclear factor-erythrocyte associated factor 2(Nrf2)and heme oxygenase-1(HO-1).The expressions of nuclear factor kappa B(NF-?B)and cyclooxygenase 2(COX-2)in myocardial tissue of mice were detected by immunohistochemical method.Results:(1)After exhaustive exercise training,the left ventricular end-systolic diameter and end-diastolic diameter increased significantly,while the ejection fraction and shortening fraction decreased significantly,indicating that the cardiac function of mice decreased after exhaustive exercise training.(2)After exhaustive exercise training,myocardial fiber swelling,myocardial fiber disarrangement,myocardial fiber rupture and inflammatory cell infiltration were observed,and the degree of myocardial fiber tissue arrangement,breakage and inflammatory cell infiltration were alleviated after CBD intervention.(3)After exhaustive exercise training,the area of blue staining area in myocardial tissue of mice was large,which indicated that there were a large number of collagen fibers depositing.After the intervention of CBD,the deposition of collagen fibers in REC group was less than that in RE group.(4)The cross-sectional area of cardiomyocytes increased significantly after exhaustive exercise training,and decreased significantly after CBD intervention,indicating that CBD can significantly inhibit cardiomyocyte hypertrophy after exhaustive exercise.(5)After exhaustive exercise training,the expression of IL-6,TNF-? and NF-?B protein increased significantly,while the expression of IL-10 protein decreased significantly.After the intervention of CBD,the expression of IL-6,TNF-? and NF-?B protein decreased significantly,while the expression of IL-10 protein increased significantly,indicating that CBD can significantly inhibit the inflammatory expression of cardiomyocytes after exhaustive exercise training.(6)The protein expression of Bax,caspase-3 and Bcl-2 increased significantly after exhaustive exercise training,while CBD significantly inhibited the expression of Bax,caspase-3 and Bcl-2 protein,indicating that CBD can significantly inhibit cardiomyocyte apoptosis after exhaustive exercise training.(7)After exhaustive exercise training,the expression of MDA-5,IRE-1?,NOX-2and COX-2 increased significantly,while the expression of SOD-1 decreased significantly,while the expression of MDA-5,IRE-1?,NOX-2 and COX-2 decreased significantly after CBD treatment,while SOD-1 increased,but there was no significant difference,indicating that CBD can significantly inhibit oxidative stress after exhaustive exercise.(8)After exhaustive exercise training,the content of Keap1 protein in myocardium increased significantly,while the protein content of Nrf2 and HO-1 in downstream nucleus decreased significantly.After CBD intervention,the transfer of Nrf2 from cytoplasm to nucleus increased significantly,transcriptional activity increased,and the expression of downstream HO-1 antioxidant protein was induced.The expression level of Keap1 protein decreased significantly,while the content of Nrf2 and HO-1 protein increased significantly,indicating that CBD may improve the cardiac function of exhaustive exercise training mice by activating Keap1/Nrf2/HO-1 signal pathway and inhibiting oxidative stress response.Conclusion:(1)CBD has a protective effect on myocardial injury in exhaustive exercise training mice,and has the effects of anti-inflammation,anti-apoptosis and antioxidant stress at the same time.(2)the protective mechanism of CBD on myocardial injury in exhaustive exercise training mice may be achieved by activating antioxidant stress Keap1/Nrf2/HO-1 signal pathway and inhibiting the expression of oxidative stress protein. |
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