| Objective To explore the effect of cis-atracurium on the diaphragm of mechanically ventilated rats and its possible molecular mechanism.MethodsPart Ⅰ18 adult male SD Rats were randomly divided into three groups:(i)the control group in which no operation was performed except fasted for 30 h;(ii)the MV group in which after 6 h of fasted,mechanical ventilation was performed for 24 h,and sodium pentobarbital(10 mg.kg-1.h-1)infusion to maintain anesthesia;(iii)the MVC group in which after 6 h of fasted,mechanical ventilation was performed for 24 h,and sodium pentobarbital(10 mg.kg-1.h-1)to maintain anesthesia and cis-atracurium to relax muscle.After mechanically ventilated for 24 h,the costal diaphragm was taken,and the crosssectional area(CSA)of the diaphragm fibers was assessed using HE staining.Anti-Slow Skeletal Myosin Heavy chain(MHC-I)was assessed using Immunochemistry and expression of two proteins related to muscle atrophy: diaphragm muscle atrophy F-box F(MAFbx),muscle specific ring finger protein 1(MURF1)was assessed using Western blotting.Part Ⅱ30 adult male SD Rats were randomly divided into five groups:(i)the control group in which no operation was performed except fasted for 30 h;(ii)the MV group in which after 6 h of fasted,mechanical ventilation was performed for 24 h,and sodium pentobarbital(10 mg.kg-1.h-1)infusion to maintain anesthesia;(iii)the MVC group in which After 6 h of fasted,mechanical ventilation was performed for 24 h,and sodium pentobarbital(10 mg.kg-1.h-1)to maintain anesthesia and cis-atracurium to relax muscle;(iv)the MVC group in which chloroquine(CQ,30 mg.kg-1)was injected i.p.24 h and 30 min prior to MV procedure and the rest of the operations were the same as the MV group;(V)the QMVC group in which CQ(30 mg.kg-1)was injected i.p.24 h and 30 min prior to MV procedure and the rest of the operations were the same as the MVC group.After mechanically ventilated for 24 h,the costal diaphragm was taken,and the CSA of the diaphragm fibers was assessed using HE staining.Expression of two proteins related to muscle atrophy was assessed using Western blotting.The localization of TOM20(mitochondrial outer membrane protein)and LC3(autophagosome protein)and the expression of LC3 was assessed using Immunofluorescence.The ultrastructure of mitochondria and autophagosomes were assessed using scanning electron microscope and the expression of mitochondrial autophagy-related proteins PTEN induced putative kinase 1(PINK1),Parkin,selective autophagy adaptor protein P62/sequestosome-1(SQSTM1)and LC3Ⅱ/Ⅰ protein using Western blotting.ResultPart Ⅰ(1)The results of arterial blood PH,PaO2 and Pa CO2 at the time of dissection of the three groups of rats were all within the normal range.There was no significant difference in the results of blood gas analysis and sodium pentobarbital dosage between the groups.(2)The CSA of the diaphragm fibers decreased significantly in the MV group compared with that in the CON group(P<0.05).And it decreased significantly in the MVC group compared with that in the CON and MV groups respectively(P<0.05).(3)The CSA of MHC-Ⅰ and MHC-Ⅱ decreased significantly in the MV group compared with that in the CON group(P<0.05),and there is no significant difference in the diaphragm fiber proportion and area fraction(P>0.05);And it decreased significantly in the MVC group compared with that in the CON and MV groups respectively(P<0.05),and there is no significant difference in the diaphragm fiber proportion and area fraction between the groups.(P>0.05).(4)The expression of MAFbx and MURF-1 proteins increased significantly in the MV group compared with that in the CON group(P<0.05).Which decreased significantly in the MVC group compared with that in the CON and MV groups respectively(P<0.05).Part Ⅱ(1)The results of arterial blood PH,PaO2 and Pa CO2 at the time of dissection of the five groups of rats were all within the normal range.There was no significant difference in the statistical results of blood gas analysis,pentobarbital sodium,and cis-atracurium dosage among the groups(P>0.05).(2)The CSA of the diaphragm fibers increased significantly in the QMV group compared with that in the MV group(P<0.05).And it increased significantly in the QMVC group compared with that in the MVC group;and compared with the CON group,there is no significant difference between QMV and QMVC groups respectively(P>0.05).(3)The expression of MAFbx and MURF-1 proteins increased significantly in the QMV group compared with that in the MV group(P<0.05).And it increased significantly in the QMVC group compared with that in the MVC group;and compared with the CON group,there is no significant difference between QMV and QMVC groups respectively(P>0.05).(4)The number of co-expressed mitochondria of TOM20 and LC3 and the expression of LC3 increased significantly in the MV group compared with those in the CON group(P<0.05).And those increased significantly in the MVC group compared with that in the CON and MV groups respectively(P<0.05).The number of co-expressed mitochondria of TOM20 and LC3 and the expression of LC3 decreased significantly in the QMV group compared with that in the MV group(P<0.05).And it decreased significantly in the QMVC group compared with that in the MVC group;and compared with the CON group,there is no significant difference between QMV and QMVC groups respectively(P>0.05).(5)It showed mild swelling of mitochondria,less autophagy and a few lipid droplets in the MV group compared with those in the CON group(P<0.05);it had more autophagy and increased mitochondrial swelling in the MVC group compared with that in the CON and MV groups respectively.It showed better mitochondrial swelling and decreased autophagy in the QMV group compared with that in the MV group and those could also be observed in the QMV group compared with that in the MV group;and compared with the CON group,there is no significant difference between QMV and QMVC groups respectively.(6)The expression of PINK1,Parkin and LC3II/I proteins increased significantly and P62 protein decreased significantly in the MV group compared with those in the CON group(P<0.05).The expression of PINK1,Parkin and LC3II/I proteins increased significantly and P62 protein decreased significantly in the MVC group compared with that in the CON and MV group respectively(P<0.05).The expression of PINK1,Parkin and LC3II/I proteins decreased significantly and P62 protein increased significantly in the QMV and QMVC groups compared with those in the MV and MVC groups respectively (P<0.05);and compared with the CON group,there is no significant difference between QMV and QMVC groups respectively.Conclusion(1)Long-term mechanical ventilation can cause diaphragm atrophy in SD rats.And both MCH-I and MCH-II fiber types have atrophied,while the diaphragm fiber proportion and area fraction do not change;(2)Cis-atracurium can aggravate diaphragm atrophy in mechanically ventilated rats may be through the AL pathway,a process that related to the PINK1/Parkin-mediated mitophagy;(3)CQ could reduce diaphragmatic atrophy caused by cis-atracurium by blocking the AL pathway. |
PDF Full Text Request