| Objective: To observe the effects of Xiao Fu Tong Jie Fang on colonic motility,enteric nervous system network structure(ENS-ICC-SMC),autophagy and apoptosis of colonic in slow transit constipation(STC)model rats,and to explore the mechanism of Xiao Fu Tong Jie Fang in the treatment of constipation from the perspective of autophagy and apoptosis regulating the homeostasis of tissue.Methods: 1.110 SD rats were randomly divided into blank group(40 rats)and modeling group(70 rats).The rat model of slow transit constipation was duplicated by three-cycle method of increasing dose of DaHuang powder suspension.Taking 10 rats in each group at the three time nodes of stool change to observe colonic pathology by HE staining,the myenteric plexus by immunofluorescence staining,the ultrastructure and autophagy of ENS-ICC-SMC by transmission electron microscopy,and to detect the expression levels of ENS-ICC related proteins PGP9.5,c-kit,Cx43,Sy,autophagy related proteins Beclin-1,LC3,p62 and apoptosis related proteins bax,bcl-2,caspase-3 in colonic myocytes by Western-Blot,apoptotic changes in colonic myocytes by TUNEL.After the model was established,the success of the model was judged according to the time of the first black stool excretion.2.Then the rest of rats were divided into blank group(group K),model group(group M),mosapride group(group S),Zhang Xichun Xiao Fu Tong Jie Decoction group(group Z)and Xiao Fu Tong Jie Fang group(group X),and given corresponding drugs for 4 weeks.To observe the body mass,the first black stool excretion time,stool weight and colon electromyography of rats in each group,Taking colon tissues to HE staining for pathology,immunofluorescence staining for Intestinal myenteric plexus,To observe ultrastructure of ENS-ICC-SMC by transmission electron microscopy,detecting the expression levels of PGP9.5,c-kit,Cx43,Sy,Beclin-1,LC3,p62,bax,bcl-2,caspase-3 and mTOR in colonic myocytes by immunofluorescence and Western-Blot,detecting the expression of Beclin-1,LC3,p62,bax,bcl-2 and caspase-3 mRNA in the colon of rats in each group by RT-PCR.Result: 1.Changes of ENS-ICC-SMC,autophagy and apoptosis in STC model rats(1)Morphological changes of ENS-ICC-SMC: With the development of modeling,HE staining showed colonic inflammatory cell infiltration,The lamina propria was swollen,the muscular fibers were necrotic,the nuclei of myocytes were pyknosis,deep staining or dissolution disappeared,The nerve cells in the myenteric plexus were vacuole degeneration and decrease.stretched preparation staining showed the distribution of colonic myenteric plexus nerves was gradually sparse,with fewer neurons and smaller fibers and cell bodies.The results of transmission electron microscopy showed that the mitochondria of rat nerve cells were swollen,even formed vacuole-like structure,occasionally see autophagic bodies and apoptotic phenomena;ICC is rare,in that its nucleus is large,chromatin edges are gathered in nucleus,a few mitochondria are in cytoplasm,mitochondria are swollen and vacuoles are formed in cytoplasm;the gap between ICC and smooth muscle cells was wider;Smooth muscle cell nucleus chromatin edge collection,a large number of vacuoles can be seen in the cytoplasm.(2)Changes of ENS-ICC related proteins: Compared with blank group,at stage 1,the expression of PGP9.5 and Cx43 tended to decrease(P>0.05),the expression of c-kit and Sy decreased(P<0.05);at stage 2 and 3,the expression of PGP9.5,c-kit,Sy and Cx43 decreased in the modeling group(P<0.05/P<0.01).(3)Changes of autophagy related proteins: Compared with the blank group,at stage 1,the expression of Beclin-1 increased,the ratio of LC3I/LC3 II decreased(P<0.05),and the expression of p62 decreased(P>0.05);at the stage 2,the expression of Beclin-1 decreased(P<0.05),the ratio of LC3I/LC3 II increased,and the expression of p62 increased(P>0.05);at the stage 3,the expression of Beclin-1 decreased,the ratio of LC3I/LC3 II increased,and the expression of p62 increased in the modeling group(P<0.01).(4)Changes of apoptosis related proteins: Compared with the blank group,at stage 1,the ratio of bcl-2/bax and caspase-3 expression did not change significantly(P>0.05);at stage 2 and 3,the ratio of bcl-2/bax decreased,and the expression of Caspase-3 increased in the model group(P<0.05/P<0.01);TUNEL showed that the apoptotic cells in colonic myometrium increased in stage 2 and 3 of modeling rats(P<0.01).2.Effect of Xiao Fu Tong Jie Fang on Body Weight,Time of First Black stool Discharge,stool volume and Colon EMG in Rats(1)Effect on body weight: Compared with group K,the weight of group M increased slowly(P<0.01);compared with group M,the weight of rats in each administration group increased faster(P<0.01).Compared with group S and group Z,the weight of rats in group X increased faster(P<0.05).(2)Effects on the first black stool and stool weight: Compared with group M,the first black stool appeared shorter and stool weigh is fewer in each administration group(P<0.01);compared with group Z,the stool weight is increased in group X(P<0.05).(3)Effect on colon electromyography: Compared with group K,colon electromyography waveform of group M was irregular,frequency slowed down and amplitude decreased(P<0.01);compared with group M,the frequency and amplitude of group S and group X increased(P<0.05/P<0.01),the frequency of group Z increased(P<0.05),amplitude trends to increase(P>0.05).3.Effect of Xiao Fu Tong Jie Fang on ENS-ICC-SMC in Colon of STC Model Rats(1)Effects on ENS-ICC-SMC morphology: HE staining showed less inflammatory infiltration,less necrosis of muscular fibers,less degeneration of myocytes and neurons of myenteric plexus,more distribution of nerve plexus,more neurons and morphological recovery,less swelling of mitochondria in neurons,ICC and SMC,more ICC and stronger cell connection in each treatment group,especially in group X.(2)Effects on ENS-ICC related proteins: The fluorescence intensity of PGP9.5,c-kit,Sy and Cx43 in group M was weaker than that in group K,while in each treatment group was stronger than in group M.;Western blotting shows the expression levels of PGP9.5,c-kit,Sy and Cx43 in group S and X increased(P<0.05/P<0.01);the expression levels of PGP9.5 in group Z showed an increasing trend(P>0.05),the expression levels of c-kit,Sy and Cx43 increased in group Z(P<0.05);compared with group Z,the expression levels of PGP9.5,Sy and c-kit in group X increased(P<0.05).4.Effect of Xiao Fu Tong Jie Fang on Autophagy and Apoptosis in STC Model Rats(1)The effect of autophagy related proteins: The fluorescence intensity of Beclin-1 and LC3 in group M was weaker than that in group K,and the fluorescence intensity of p62 was stronger than that in group K.The fluorescence intensity of Beclin-1 and LC3 in each treatment group was stronger than that in group M,and the fluorescence intensity of p62 was weaker than that in group M.;Western blotting shows Beclin-1 expression increased in group S and X(P<0.05/P<0.01),p62 expression decreased in group Z and X(P<0.05/P<0.01),and LC3 I/II ratio decreased in group Z and X(P<0.05);Compared with group Z,the expression of Beclin-1 increased and p62 decreased in group X(P<0.05);Compared with group S,the expression of p62 in group X decreased(P<0.05).(2)Effect of autophagy related genes: Compared with group K,the expression of Beclin-1,LC3 and p62 in group M decreased(P<0.01);compared with group M,the expression of Beclin-1 in group S and X increased(P<0.05/P<0.01);compared with group Z,the expression of Beclin-1 in group X increased(P<0.05),while the expression of LC3 and p62 in each group did not change significantly(P>0.05).(3)The effect on apoptosis related proteins: Compared with group M,the the fluorescence staining of bcl-2,bax and caspase-3 in group decreased in each group;Western blotting shows the ratio of bcl-2/bax in group S and X increased(P<0.01),the expression of caspase-3 decreased in group S and X(P<0.05/P<0.01),the ratio of bcl-2/bax trend to increased and the expression of caspase-3 trend to decreased In group Z(P>0.05);Compared with group Z,the ratio of bcl-2/bax in group X increased(P<0.05).(4)Effect on apoptosis related genes: Compared with group K,the expressions of bcl-2,bax and caspase-3 in group M increased(P<0.01);compared with group M,the expression of bcl-2 trend to decrease in all groups(P>0.05),the expression of bax decreased in all groups(P<0.05);The expression of caspase-3 in group Z trend to decrease(P>0.05),while the expression of caspase-3 in group S and group X decreased(P<0.05).(5)Effect on mTOR: The fluorescence in group M was stronger than that in group K,and the fluorescence of each treatment group was weaker than that in group M.Western blotting shows that Compared with group K,the expression of p-mTOR increased and the ratio of p-mTOR/mTOR increased in group M(P<0.01);compared with group M,the ratio of p-mTOR/mTOR trend to decrease in group S and Z(P>0.05),the expression of p-mTOR decreased in group X,and the ratio of p-mTOR/mTOR decreased(P<0.05).Conclusion: 1.Dynamics disorder of colon in STC model rats is related to damage of intestinal nervous system network structure.Abnormal autophagy and apoptosis may participate in the process of damage of intestinal nervous system network structure in slow transit constipation model rats.2.Xiao Fu Tong Jie Fang can improve the constipation symptoms of slow transit constipation model rats,improve the frequency and amplitude of colon electromyography,promote the repair of ENS-ICC-SMC and increase the expression levels of PGP9.5,c-kit,Sy and Cx43.3.Xiao Fu Tong Jie Fang may increase the expression of Beclin-1,decrease the ratio of LC3 I/II,decrease the expression of p62,enhance the activity of autophagy,decrease the expression of bax,increase the ratio of bcl-2/bax,decrease the expression of Caspase-3 and reduce apoptosis,by inhibiting the phosphorylation of mTOR in colonic muscles.4.Xiao Fu Tong Jie Fang may regulate autophagy and apoptosis of colon cells,promote the repair of enteric nervous system network structure,correct colonic motility disorder in STC rats,and tonify kidney and enrich essence method plays an important role in it. |
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