| Objective The model of gastric emptying disorder in mice was established by using L-arginine,a precursor of nitric oxide(NO),and curcumin was given to the stomach in advance to observe whether curcumin alleviated gastric emptying disorder in mice was related to the signaling pathway of Interstitial Cells of Cajal(ICC)which initiated slow wave peristalsis in gastric tissue.The mechanism of curcumin action was studied by detecting the changes of ICC-specific tyrosine kinase receptor(c-kit)and its specific ligand(SCF),calcium-activated chloride channel functional protein(Anoctamin-1,Ano1)and gap junction protein 43(connexin 43,Cx43)between ICC and smooth muscle cells.Methods Thirty-six 4-week-old healthy Kunming male mice weighing 22-25 g were randomly divided into control group,curcumin group,L-arginine group and curcumin+L-arginine group after one week of adaptive feeding,with 9 mice in each group.The curcumin group and curcumin+L-arginine group were given 0.2 ml curcumin suspension(200 mg/kg)by gavage once a day for 15 consecutive days.The other groups were given the same amount of solvent solution by gavage as control group.The mice in L-arginine group and curcumin+L-arginine group were given 0.2 ml L-arginine(2 g/kg)suspension respectively for 5 days from the 11 th day of gastric administration.After the experiment,the mice were fasting for 24 hours and could drink water freely.Before the gastric emptying rate was determined,each mouse was given 0.8 ml of Arabic gum suspension once.After 20 minutes,the mice were killed by dislocation.The gastric emptying rate was measured in each group.The expression of c-kit,SCF,Ano1 and Cx43 were detected by real-time quantitative RT-PCR.The expression of c-kit,Ano1 and Cx43 at protein level was detected by Western blot.The expression of c-kit,Ano1 and Cx43 was detected by immunofluorescence.Results The gastric emptying rate of L-arginine group(32.83±11.20)was significantly lower than that of the control group(56.67±9.18)(P<0.01).The expression of c-kit(0.62±0.22)and SCF(0.58±0.19)m RNA in L-arginine group were significantly lower than that in control group(P<0.01).The changes of c-kit protein in gastric tissues detected by Western blot were basically the same as those of the above-mentioned m RNA expression.Compared with the control group,the immunohistochemical staining results of frozen sections of gastric tissues in L-arginine group showed that the positive expression of c-kit was mainly located between the circular and longitudinal smooth muscle of gastric wall,and the amount and intensity of the positive expression were similar to those detected by Western blot.The expression of Ano1 m RNA in L-arginine group(0.51±0.23)was lower than that in control group(P<0.01).The changes of Ano1 protein in gastric tissue detected by Western blot were basically consistent with those of the above-mentioned m RNA expression.Immunohistochemical staining showed that the positive expression of Ano1 was mainly located between the circular and longitudinal smooth muscle of gastric wall.The quantity and intensity of the positive expression were similar to those detected by Western blot.The expression of Cx43 m RNA in L-arginine group(0.55±0.15)was significantly lower than that in control group(P<0.01).The changes of Cx43 protein and in gastric tissues detected by Western blot were basically the same as m RNA.The positive expression of Cx43 was widely distributed around smooth muscle cells.The positive staining proportion did not change significantly,but the average staining intensity was similar to that of Western blot.Compared with L-arginine group,the gastric emptying rate of mice in curcumin+L-arginine group was significantly increased(48.39±10.34)(P<0.01);the expression of c-kit(0.83±0.19)(P<0.05)and SCF(0.86±0.15)(P<0.01)m RNA in gastric tissues was significantly improved.The expression of c-kit protein was increased by Western blot,and the expression of immunohistochemical positive protein was also increased.The expression of Ano1 m RNA was significantly improved(0.87+0.20)(P<0.05).The expression of Ano1 protein was increased by Western blot and the expression of immunohistochemical positive protein was also increased.The expression of Cx43 m RNA was significantly improved(0.81 +0.20)(P<0.05).The expression of Cx43 protein was increased by Western blot,and the proportion of positive staining of immunofluorescence expression was not changed,but the average staining intensity was slightly increased.Conclusions Curcumin can significantly improve functional gastric emptying disorder caused by exogenous nitric oxide,and its mechanism is related to the improvement of ICC-related signaling pathways in gastric tissue.Curcumin may restore the slow-wave initiation function of ICC by increasing the expression of c-kit/SCF,Ano1 and Cx43 in gastric tissue of mice,improve the information transmission between ICC and smooth muscle cells,and thus improve the gastrointestinal dysfunction caused by exogenous nitric oxide. |
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