Effect of PKC-mTOR pathway on apoptosis of gastric smooth muscle cells in diabetic rats and its mechanism
AIM:To investigate the effect of protein kinase C(PKC)-mammalian target of rapamycin(mTOR)pathway on the apoptosis of gastric smooth muscle cells in diabetic rats,and to explore the related mechanism.METHODS:Male and female 4-week-old specific pathogen-free Sprague-Dawley rats were randomly allocated to control,diabetes,diabetes+0.1 µmol/L phorbol 12,13-myristate acetate(PMA),diabetes+0.2 µmol/L PMA,diabetes+0.5 µmol/L PMA,diabetes+2 µmol/L bisindolylmaleimide I(GF109203X),diabetes+5 µmol/L GF109203X,and diabetes+10 µmol/L GF109203X groups(n=9).Primary rat gastric smooth muscle cells cultured in a high-glucose environment in vitro were treated with 0.1 µmol/L PMA,0.2 µmol/L PMA,0.5 µmol/L PMA,2 µmol/L GF109203X,5 µmol/L GF109203X,10 µmol/L GF109203X,or vehicle.The isolated muscle technique was used to evaluate the spontaneous contraction of gastric antral smooth muscle.The pathologic changes and apoptosis of rat gastric smooth muscle were identi-fied using HE staining and flow cytometry,respectively.The expression levels of stem cell factor(SCF),c-Kit,mTOR,phosphorylated(p)-mTOR,p70 ribosomal protein S6 kinase(p70S6K),p-p70S6K,eIF4E-binding protein 1(4E-BP1),p-4E-BP1,caspase-3,B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)in gastric smooth muscle were measured by Western blot.RESULTS:The contractility of the gastric antral smooth muscle was lower(P<0.05),gastric smooth muscle atrophy and the apoptosis rate was higher(P<0.01),and the expression of SCF,c-Kit,and p-mTOR was lower(P<0.05 or P<0.01)in the diabetes group than in the control group.There was no significant difference in the spon-taneous contraction of the gastric antral smooth muscle between the diabetes and diabetes+0.1 µmol/L PMA groups,but it was significantly greater in the diabetes+0.2 µmol/L PMA and diabetes+0.5 µmol/L PMA groups(P<0.05 or P<0.01).The phosphorylation levels of mTOR,p70S6K(T421/S424),and p70S6K(T389)were also higher in the diabetes+0.2 µmol/L PMA and diabetes+0.5 µmol/L PMA groups than in diabetes group(P<0.01).Furthermore,the phosphorylation of 4E-BP1 was higher(P<0.05)in the diabetes+0.5 µmol/L PMA group than in the diabetes group(P<0.01).There was no significant difference in the spontaneous contraction of the gastric antral smooth muscle between the diabetes and diabe-tes+2 µmol/L GF109203X groups,but it was significantly lower in the diabetes+5 µmol/L GF109203X and diabetes+10 µmol/L GF109203X groups(P<0.05 or P<0.01).The phosphorylation levels of mTOR and p70S6K(T389)were lower in the diabetes+5 µmol/L GF109203X and diabetes+10 µmol/L GF109203X groups(P<0.05),the phosphorylation of p70S6K(T421/S424)was lower in the diabetes+10 µmol/L GF109203X group(P<0.05),and the phosphorylation of 4E-BP1 was lower in the diabetes+GF109203X(2,5 and 10 µmol/L)groups(P<0.05)than in the diabetes group.Com-pared with the CK group,the apoptosis rate was high in the GF109203X-treated(2,5 and 10 µmol/L)groups(P<0.01),the expression of Bax and caspase-3 was low(P<0.05),and the expression of Bcl-2 was high in the PMA-treated(0.1,0.2 and 0.5 µmol/L)groups(P<0.05).CONCLUSION:The downregulation of the PKC-mTOR pathway in the gastric smooth muscle of diabetic rats leads to apoptosis of gastric smooth muscle cells through Bax/Bcl-2 and caspase-3 and the downregulation of SCF and c-Kit,which may play an important role in the gastric dysmotility that characterizes diabetes.
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