| Part I The Effect of Cigarette Smoke on Skeletal Muscle Atrophy and Senescence with Histone Deacetylase 2 in Skeletal Muscle Objective: To explore the effects of cigarette smoke(CS)on skeletal muscle atrophy and senescence as well as its effect on histone deacetylase 2(HDAC2).Method:(1)Healthy adult clean-grade male C57BL/6 mice were used as experimental subjects,and mice(3-4 weeks old,14±2 g body weight)were randomly divided into 4 groups of 10 mice each using the random number method,namely,12-week control group(N-12W),12-week cigarette smoke exposure group(CS-12W),24-week control group(N-24W),24-week cigarette smoke exposure group(CS-24W).Among them,the CS-12 W and CS-24 W groups were given 12 weeks and 24 weeks of cigarette smoke stimulation,respectively,and the N-12 W and N-24 W groups were given the same duration of air exposure,respectively.The general condition and weight changes of mice in each group were observed regularly.After the end of cigarette smoke stimulation,mice in each group were executed,lung tissues and gastrocnemius muscles in each group were kept,and the wet weight of one side of gastrocnemius muscle was weighed,and the morphological changes of lung and skeletal muscles were observed by HE staining,and the protein expression levels of HDAC2,MURF1,MAFbx,P53,P21 and SMP30 in each group were detected by Western blot.The m RNA expression levels of HDAC2 were detected by fluorescence quantitative PCR.(2)C2C12 mouse myogenic cells were used as the research object,and mature skeletal muscle cells were obtained by differentiation induced with 2% horse serum for 6 days.Firstly,the protein expression of HDAC2,MURF1,MAFbx and myosin heavy chain(MHC)myotube diameter changes were detected by Western Blot and immunofluorescence after stimulating skeletal muscle cells with different CSE concentrations(0,0.01%,0.1%,0.2%,0.3%)for 24 hours.Result:(1)Compared with the control groups(N-12 W,-24W),the body weight and gastrocnemius muscle mass of mice in the CS group were significantly decreased and further reduced with increasing smoking time(P<0.05);the alveolar septum and alveolar wall were thickened and the mean lung lining interval(MLI)was increased in the CS group and further increased with increasing smoking time(P<0.05);the cross-sectional area of gastrocnemius muscle of mice in the CS group(CSA)was significantly reduced and further decreased with the increase of smoking time(P<0.05).(2)The expression of HDAC2 protein and m RNA in the gastrocnemius muscle of CS group mice was decreased compared with the control group,and the expression level was further reduced with the increase of smoking time(P<0.05).In contrast,the protein expression of MURF1,MAFbx,P53,and P21 increased and further increased with the increase of smoking time(P<0.05).In addition,the protein expression of senescence marker protein SMP30 decreased and further decreased with the increase of smoking time(P<0.05).(3)Compared with the control group(N),myotube diameter was significantly reduced at CSE concentrations of 0.1%,0.2%,and 0.3% after 24 hours of CSE stimulation(P<0.05);HDAC2 protein expression decreased at CSE concentrations of 0.1%,0.2%,and 0.3% compared with the control group(N)and was concentration-dependent(P<0.05);Compared with the control group(N),HDAC2 protein expression decreased at CSE concentrations of 0.1%,0.2% and 0.3% with concentration dependence(P<0.05),while MAFbx and MURF1 protein expression increased at CSE concentrations of 0.1%,0.2% and 0.3% with concentration dependence(P<0.05).Conclusion: CS can cause emphysema along with the development of skeletal muscle atrophy and senescence,a process that may be associated with the downregulation of HDAC2 expressionPart II The role of histone deacetylase 2 in cigarette smoke extract-inducedatrophy and senescence in C2C12 cellsObjective: To explore the role of HDAC2 in cigarette smoke-induced skeletal muscle atrophy and senescence,and to observe the effect of HDAC2 on skeletal muscle atrophy and senescence through the regulation of NF-κB signaling pathway.Method:(1)The C2C12 cells were studied and mature skeletal muscle cells were obtained by induction of differentiation with 2% horse serum for 6 days followed by addition of 0.2% CSE and continued stimulation for 24 hours.Normal negative control(LV-NC),normal+HDAC2 overexpression(LV-HDAC2),CSE+negative control(CSE+LV-NC),and CSE+HDAC2 overexpression group(CSE+LV-HDAC2)were set up in the experiment,and the transfection after lentivirus overexpression of HDAC2 was firstly detected by Western Blot,fluorescence quantitative PCR and overexpression efficiency;then the changes of myotube diameter in each group were detected by immunofluorescence,and the expression levels of atrophy-related markers MURF1,MAFbx and senescence-related markers P53,P21,SMP30,NF-κB p65,IKK protein and m RNA were detected by Western Blot,fluorescence quantitative PCR,and β-galactosidase staining to detect cellular senescence changes;(2)Skeletal muscle cells were preincubated with the NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)for 2 hours,followed by stimulation with 0.2% CSE for 24 hours.Groupings were set according to the presence or absence of HDAC2 or PDTC overexpression intervention: CSE,CSE+LV-HDAC2,CSE+PDTC,CSE+LV-HDAC2+PTDC groups.Immunofluorescence was used to detect changes in myotube diameter in each group,and Western Blot was used to detect the expression levels of NF-κB p65,MURF1,MAFbx,P53,and P21 proteins in each group.Result:(1)HDAC2 protein and m RNA expression were significantly increased in C2C12 cells after lentiviral overexpression of HDAC2(P<0.05);Then C2C12 cells were stimulated with the addition of 0.2% CSE for 24 hours.Compared with the normal negative group(LV-NC),myotube diameter was significantly reduced in the LV-NC and LV-HDAC2 groups under CSE stimulation(P<0.05);and Myotube diameter increased in the CSE+LV-HDAC2 group compared with the CSE+LV-NC group(P<0.05).Meanwhile,the protein and m RNA expressions of atrophy-related markers MURF1 and MAFbx were significantly higher in the CSE-stimulated LV-NC and LV-HDAC2 groups than in the control LV-NC group,while the protein and m RNA expressions of MURF1 and MAFbx were lower in the CSE+LV-HDAC2 group than in the CSE+LV-NC group(P<0.05).(2)Compared with the normal negative group(LV-NC),senescent cells were significantly increased in the CSE-stimulated LV-NC group and LV-HDAC2 group(P<0.05),while senescent cells were reduced in the CSE+LV-HDAC2 group compared with the CSE+LV-NC group,but still higher than the control LV-NC group(P<0.05);meanwhile,the CSE-stimulated LV-NC group and The expression of senescence-related markers P53,P21 protein and m RNA were significantly increased in the LV-NC group and LV-HDAC2 group compared with the control LV-NC group,while the expression of P53,P21 protein and m RNA were lower in the CSE+LV-HDAC2 group than in the CSE+LV-NC group,in addition,SMP30 was expressed in contrast to P53 and P21 protein,and the results were statistically significant(P<0.05).(3)Compared with the normal negative group(LV-NC),NF-κB p65 and IKK protein expressions were significantly increased in the CSE+LV-NC and CSE+LV-HDAC2 groups(P<0.05);Compared with the CSE+LV-NC group,NF-κB p65 and IKK protein expressions were decreased in the CSE+LV-HDAC2 group(P<0.05).(4)Compared with the CSE group,NF-κB p65 protein expression was decreased after overexpression of HDAC2 alone or addition of PDTC under CSE stimulation(P<0.05),while NF-κB p65 protein expression was decreased after overexpression of HDAC2 with addition of PDTC compared with HDAC2 alone or addition of PDTC(P<0.05).(5)Compared with the CSE group,the myotube diameter of cells overexpressing HDAC2 alone or adding PDTC increased after CSE stimulation(P<0.05);while the myotube diameter of cells overexpressing HDAC2 and adding PDTC increased compared with HDAC2 alone or adding PDTC group(P<0.05).(6)Compared with the CSE group,the expression of atrophy-related markers MURF1 and MAFbx and senescence-related markers P53 and P21 were decreased after overexpression of HDAC2 alone or addition of PDTC under CSE stimulation(P<0.05);While the overexpression of HDAC2 with the addition of PDTC decreased compared with the overexpression of HDAC2 alone or addition of PDTC in the MURF1,MAFbx and P53,P21 protein expression groups(P<0.05).MAFbx and P53,P21 protein expression were reduced in the HDAC2 overexpression group compared with HDAC2 alone or PDTC(P<0.05).Conclusion: HDAC2 plays an important role in CSE-induced skeletal muscle cell atrophy and senescence,and overexpression of HDAC2 inhibits skeletal muscle atrophy and senescence.Meanwhile,HDAC2 inhibited cigarette smoke-induced skeletal muscle cells atrophy and senescence through mediating NF-κB signaling pathway.Part III The Effect and Mechanism of Resveratrol on Skeletal Muscle Atrophy and Senescence Induced by Cigarette SmokeObjective: To observe the effect of resveratrol(RSV)intervention on cigarette smoke-induced skeletal muscle atrophy and senescence,and to investigate whether HDAC2 has an important role in the improvement of skeletal muscle atrophy and senescence caused by RSV.Method:(1)Healthy adult clean grade male C57BL/6 mice were used as experimental subjects,and the mice were divided into 4 groups of 10 mice each by random number method,which were control group(N),resveratrol intervention group(RSV),24-week cigarette smoke exposure group(COPD),and cigarette exposure+resveratrol intervention group(C+RSV).The CS and C+RSV groups were exposed to cigarette smoke for 24 weeks,and the C+RSV group was given RSV(200 mg/kg)by gavage at 20 weeks;the N and RSV groups were given the same duration of air exposure,and the RSV group was also given RSV(200 mg/kg)by gavage.After the cigarette smoke intervention,the mice in each group were executed,and the lung tissue and gastrocnemius muscle of each group were kept,and the wet weight of one side of the gastrocnemius muscle was weighed,and the morphological changes of lung and skeletal muscle were observed by HE staining.The protein expression levels of HDAC2,MURF1,MAFbx,P53,P21,SMP30 and IKK,NF-κBp65 were detected by Western blot.(2)C2C12 mouse myogenic cells were studied,and mature skeletal muscle cells were obtained by inducing differentiation with 2% horse serum for 6 days after preincubation with 50 u M RSV for 24 hours,followed by stimulation with 0.2% CSE for 24 hours.The cells were first divided into four groups: normal control group(N),cigarette extract stimulation group(CSE),resveratrol group(RSV),and resveratrol+cigarette extract stimulation group(CSE+RSV),and the myotube diameters of each group were detected using immunofluorescence,and Western blot was used to detect HDAC2,MURF1,MAFbx,P53,P21,IKK,NF-κBp65 protein expression levels,fluorescence quantitative PCR for HDAC2,MURF1,MAFbx m RNA expression levels,Elisa for inflammatory factors IL-1β,TNF-α concentrations;then Western Blot and fluorescence quantitative PCR were used to detect transfection and overexpression efficiency after lentiviral down-expression of HDAC2.Subsequently,CSE and RSV were added and cells were divided into four groups: cigarette extract stimulation group(CSE),resveratrol+cigarette extract stimulation group(CSE+RSV),resveratrol+cigarette extract stimulation group+null lentivirus group(CSE+RSV+Si RNA-NC),resveratrol+cigarette extract stimulation group+lowered expression HDAC2 group(CSE+RSV+ The protein expression levels of MURF1,MAFbx,P53,P21 and SMP30 were detected by Western blot,and the concentrations of inflammatory factors IL-1β and TNF-α were detected by Elisa.Result:(1)Compared with the cigarette smoke exposed group(COPD),no significant changes in body weight and gastrocnemius weight were observed in the C+RSV group(P>0.05),while alveolar septum and alveolar wall were thinner and mean lung lining interval(MLI)was reduced(P<0.05),and accompanied by an increase in gastrocnemius cross-sectional area(CSA)(P<0.05);compared with the cigarette smoke exposed group(CS),the expression of HDAC2 protein and m RNA in the gastrocnemius of the C+RSV The expression of HDAC2 protein and m RNA was increased in the gastrocnemius muscle(P<0.05),while the expression of MURF1,MAFbx,P53,P21,IKK,and NF-κB p65 protein was decreased in the CS+RSV group compared with the COPD group(P<0.05);meanwhile,the concentrations of IL-1β and TNF-α were significantly decreased in the gastrocnemius muscle of the CS+RSV group compared with the COPD group(P<0.05).(2)Compared with the normal control group(N),the cellular myotubular diameter was reduced in the CSE group and CSE+RSV group(P<0.05);compared with the CSE group,the cellular myotubular diameter was increased in the CSE+RSV group(P<0.05).Meanwhile,the expression of atrophy markers MURF1,MAFbx protein and m RNA were increased in CSE group and CSE+RSV group(P<0.05);while the expression of MURF1,MAFbx protein and m RNA were decreased in CSE group after RSV intervention(P<0.05).(3)Compared with the normal control group(N),the expression of aging markers P53 and P21 increased in CSE group and CSE+RSV group(P<0.05);while the expression of P53 and P21 in CSE group could be reduced after RSV intervention(P<0.05).(4)HDAC2 protein and m RNA expression were increased in the CSE group and CSE+RSV group compared with the normal control group(N)(P<0.05);while HDAC2 protein and m RNA expression in the CSE group could be reduced after RSV intervention(P<0.05).(5)Compared with the normal control group(N),IKK and NF-κB p65 protein expression and inflammatory factors IL-1β and TNF-α concentrations were increased in the CSE group and CSE+RSV group(P<0.05);while RSV intervention could reduce IKK and NF-κB p65 protein expression and inflammatory factors IL-1β and TNF-α concentrations in the CSE group(P <0.05).(6)HDAC2 protein and m RNA expression were significantly reduced in C2C12 cells after lentiviral down-expression of HDAC2(P<0.05);Then the addition of RSV and 0.2% CSE stimulated C2C12 cells for 24 hours.Compared with the CSE group,the CSE+RSV group,CSE+RSV+Si RNA-NC group, CSE+RSV+Si RNA-HDAC2 group had decreased expression of atrophy markers MURF1 and MAFbx and senescence markers P53 and P21 proteins(P<0.05);Compared with CSE+RSV+Si RNA-NC,CSE+RSV+Si RNA-HDAC2 group had increased expression of MURF1,MAFbx and P53 and P21 proteins(P<0.05);and senescence marker protein SMP30 had the opposite trend with P53 and P21 proteins,and the results were statistically significant(P<0.05).(7)Compared with the CSE group,the concentrations of inflammatory factors IL-1β and TNF-α were reduced in the CSE+RSV,CSE+RSV+Si RNA-NC and CSE+RSV+Si RNA-HDAC2 group(P<0.05);Compared with the CSE+RSV+Si RNA-NC group,the inflammatory factor IL-1β,TNF-α concentrations were increased in the CSE+RSV+Si RNA-NC group compared with the CSE+RSV+HDAC2 group(P<0.05).Conclusion: RSV not only ameliorates the formation of emphysema caused by cigarette smoke,but also has the effect of inhibiting the onset of skeletal muscle atrophy and senescence.This process may be related to its upregulation of HDAC2 and inhibition of inflammatory responses due to NF-κB signaling pathway activation. |
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