| Objective: To study and analyze the effect of Zhishi on endoplasmic reticulum stress-autophagy response within interstitial cells of Cajal(ICCs)and further clarify the mechanism of Zhishi regulating ERS-autophagy response based on the IRE1-TRAF2-JNK-Beclin1 signal transduction pathway.Methods: The ICCs in FD rats and isolated ICCs were selected as the research object.(1)FD model was induced by chronic stress via tail clamping.48 SD rats were randomly divided into control group,model group,zhishi group and duopanlitong group(each n=12),then fed the rats with corresponding drugs.The gastric emptying rates were measured.The expression changes of GRP78(ERS biomarker),LC3B(autophagy biomarker)were detected by In situ hybridization and Western blot(WB).(2)ICCs were isolated by collagenase type II digestion and identified by immunofluorescence staining.Tunicamycin(TM)was used to induce ERS model,the SP600125 was used to block ERS.The zhishi-serum was prepared according to serum pharmacology on SD rats.Then the ICCs were randomly divided into 4 groups and received different interventions,including control group(normal culture),ERS group(ICCs were stimulated with 2?g/ml TM),zhishi group(ICCs were co-stimulated with 2?g/ml TM and 10% zhishi-serum)and SP600125 group(ICCs were co-stimulated with 2?g/ml TM and 10?mol/l SP600125),the ICCs in all groups were treated for 6 hours.The endoplasmic reticulum ultrastructure and autophagosomes of ICCs were examined using transmission electron microscope.ICCs activity was detected by CCK-8 assay.The expressions of IRE1,TRAF2,JNK and Beclin1 were detected by qRT-PCR and WB.Results:(1)The FD model was successfully induced by chronic stress via tail clamping.Compared with controls,the gastric emptying rate was decreased in model group(P<0.05),compared with models,the gastric emptying rates were increased in zhishi and duopanlitong group(P<0.05).ISH studies showed significant quantities of ICCs in the gastric myenteric plexus,increased GRP78 and LC3 B in model group compared with controls(P<0.05),and decreased GRP78 and LC3 B in zhishi and duopanlitong group compared with models(P<0.05).Moreover,Western blot confirmed GRP78 and LC3 B protein was greater in FD rats compared with controls were increased(P<0.05).Compared with models,the expressions of GRP78,LC3 B were decreased in zhishi and duopanlitong group(P<0.05).(2)Succeeded in ICCs isolating and identifying.In controls,the ultrastructures of ICCs were normal with abundant rough ER and few autophagosomes.Altered ICCs ultrastructures were observed in model group as was expanded rough ER,even degranulation,and a large number of autophagosomes,while a small amount of degranulation,less autophagosomes in zhishi and SP600125 group.(3)Compared with the control group,the level of ICCs activity was decreased in ERS group(P<0.05).Zhishi and SP600125 group can improve the growth activity of ICCs when compared with ERS group(P<0.05).(4)Compared with the control group,the mRNA expressions of IRE1,TRAF2,JNK and Beclin1 were increased in ERS group(P<0.05).Compared with the ERS group,decreases in IRE1,TRAF2,JNK,Beclin1 were observed in zhishi group and SP600125 group(P<0.05).Compared with the zhishi group,the IRE1 increased but opposite trends were observed for JNK,Beclin1 in SP600125 group(P<0.05).(4)Compared with the control group,the protein expressions of IRE1,TRAF2,JNK and Beclin1 were increased in ERS group(P<0.05).Compared with the ERS group,decreases in IRE1,TRAF2,JNK,Beclin1 were observed in zhishi group(P<0.05),increases in IRE1 and decreases in TRAF2,JNK,Beclin1 were observed in SP600125 group.Compared with the zhishi group,the IRE1,TRAF2,JNK and Beclin1 were increased in SP600125 group(P<0.05).Conclusions: The gastric motility disorder in FD rats can be induced by chronic stress via tail clamping with the occurrence of ICCs ERS-autophagy response.Zhishi can reduce the ERS-autophagy injury to regulate the gastric motility,and the mechanism might be related to the IRE1-TRAF2-JNK-Beclin1 signal transduction pathway. |
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