| Background and Aim Myopathy is a clinical disease in which skeletal muscle function is abnormal.The decline of skeletal muscle regeneration ability is a key factor of myopathy deterioration.Studies have shown that andrographolide,the main effective component of Chinese traditional medicine Andrographis paniculata,improves the pathological symptoms of skeletal muscle in MDX mice.However,the effect of andrographolide on skeletal muscle regeneration remains unclear.The aim of this study is to explore the effect and mechanism of andrographolide on skeletal muscle regeneration,which would provide theoretical basis for further development of andrographolide in clinical applications.Methods Part I: 1.The effect of andrographolide on histopathology of mouse skeletal muscle regeneration.C57/BL6 mice were randomly divided into sham operation group,model group and andrographolide group,respectively.Mice were treated with andrographolide(10 mg/kg)by intraperitoneal injection for 7 consecutive days,20μl Cardiotoxin(10μM)was injected into the anterior tibial muscle of the hindlimb of mice to establish the acute injury model of skeletal muscle,and the sham operation group was injected with the same volume of saline.The animals were consecutively treated with andrographolide for 3 weeks and the tibialis anterior muscle was harvested at different time point.The samples were fixed with 4% paraformaldehyde and paraffin-embedded was performed.H&E staining was performed and the images were collected using optical microscope.The average number,cross sectional diameter and area of new muscle fibers were analyzed.2.The effect of andrographolide on proliferation and migration of Myoblast. The mouse satellite cells were isolated and cultured.The activity of satellite cells and C2C12 mouse myoblasts treated with different concentrations of andrographolide were detected by CCK-8 kit.Satellite cells and C2C12 cells were treated with andrographolide and cell count experiment was performed.The expression level of PCNA protein of C2C12 was detected by using Western Blot.Real-time PCR was performed to detect the transcription levels of cell cycle genes in mouse satellite cells.The number of positive cells expressing Ki67 and PCNA protein was detected by immunohistochemistry.The effect of andrographolide on migration of satellite cells and C2C12 cells was detected by cell scratch assay.3.The effect of andrographolide on differentiation and fusion of Myoblast.The satellite cells and C2C12 cells were cultured in medium containing 10%FBS and induced differentiation by 2% Donor Equine Serum.The transcription level of Myogenic gene of andrographolide treated cells was detected by Real Time-PCR.The expression levels of Myo D and Myosin in the regenerated muscle fibers of mice were detected by immunohistochemistry.C2C12 cells were induced to differentiate into myotubules,and the number of myotubules were counted.The expression of Myosin protein was detected by immunofluorescence staining.Part Ⅱ: 1.The effect of andrographolide on Histone methylation of Myoblast.Western Blot detected the methylation levels of histone H3K4 of C2C12 cells treated with different concentrations of andrographolide and satellite cells treated andrographolide at different time point.2.The effect of andrographolide on differentiation and fusion of Myoblast after Histone methylation inhibition The expression level of H3K4Me2/3 in mouse regenerated muscle fibers was detected by immunohistochemistry.Mouse satellite cells and C2C12 cells treated with andrographolide,followed by DZNe P treatment.Real Time-PCR was performed to detect the differentiation and fusion gene.The C2C12 cells was induced to myotube by 2% Donor Equine Serum,followed by DZNe P and andrographolide treatment.The number of myotubules was observed and counted after crystal violet staining.Results: Part I: 1.Andrographolide promotes muscle skeletal muscle regeneration.H&E staining results showed that inflammatory cell recruitment and myolysis at the first day after modeling.At day 3,extracellular matrix remodeling was exhibited,whereas well-organized extracellular matrix was observed in Andrographolide treated group.At day 7,new muscle fibers with central nucleus were observed,and the number of integrate regenerated myofibers in the andrographolide group was more than that in the model group.At day 21,the restoration of damaged muscle fibers was basically completed,and the cross-sectional diameter and area of regenerated myofibers in the andrographolide group were better than those in the model group.2.Andrographolide was not effect on the proliferation and migration of Myoblast CCK-8 results showed that the cell activity decreased when the concentration of andrographolide was greater than 1μM.Compared with the control group,the number of satellite cell proliferation in the andrographolide group was not significantly difference.Western Blot assay showed that andrographolide had no significant effect on the expression of PCNA protein in C2C12 cells.Real-time PCR,immunohistochemistry and immunofluorescence results showed that there were no significant differences in the transcription levels of cell cycle regulation genes and the expression of PCNA,Ki67 proteins between the andrographolide group and control group.3.Andrographolide promotes the differentiation and fusion of Myoblast.Real time-PCR analysis showed that the transcription level of differentiation gene in andrographolide group was higher than that in control group,and the number of myotubules formed in C2C12 cells increased after andrographolide treatment.Immunohistochemical results also indicated that the expression levels of Myo D and Myosin in the andrographolide group were increased compared with that in the control group.Part Ⅱ: 1.Andrographolide up-regulates the methylation level of H3K4 of Myoblast Western Blot results showed that the methylation levels of H3K4 in the andrographolide group were higher than those in the control group.The expression of H3K4Me2/3 was increased with the prolonging of treatment time and was concentration-dependent.2.Andrographolide can promote differentiation and fusion of Myoblast through H3K4 methylation pathway Real time-PCR results showed that the transcription level of differentiation genes decreased in the andrographolide group after inhibiting histone methylation with DZNe P,and the results of C2C12 induced differentiation into myotube experiment showed that the number of myotubule in the andrographolide group decreased after inhibiting histone methylation.Conclusion: The results of this study indicate that andrographolide can promote the regeneration of skeletal muscle in mice,and the mechanism may be that andrographolide up-regulates the transcription of myogenic genes by regulating histone methylation. |
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