| Study background:Diabetic gastroparesis(DGP),the most common gastrointestinal tract complication of diabetes,is characterized by decreased gastrointestinal motility and delayed gastric emptying without mechanical obstruction.However,the pathogenesis of DGP remains unclear.Currently,the presence of autonomic neuropathy,acute elevations in blood glucose concentrations,and loss or injury of interstitial cells of Cajal are considered to be key risk factors for delayed gastric emptying.IGF-1 is mainly secreted by the liver and is a protein peptide with a certain similarity in structure to insulin.In recent years,many studies have found that IGF-1 is closely related to the occurrence and development of chronic complications of diabetes.The phosphoinositide 3-kimase(PI3K)-Akt pathway is an essential intracellular signaling pathway in cell cycle.The PI3K-Akt pathway is a downstream of IGF-1,which is activated in many diabetic complications.Zhang et al.found that the PI3K-Akt pathway is involved in DGP development,and PI3K-Akt expression gradually decreases with the development of gastroparesis.Protein kinase C(PKC)family is classified into three subfamilies,including classical,novel and atypical PKCs,classical PKCs include PKCa,PKC??,PKC??,and PKC?.Classical PKC,also known as Ca2+-dependent PKC,is downstream signal of PI3K-Akt pathway.Classical PKCs have a typical structure of Ca2+binding domain(C2 domain),which require both Diacylglycerol(DG)and Ca2+ for activation.PKC is also involved in the regulation of Ca2+ channel opening,affects the influx of extracellular Ca2+,and has a major influence on the changes in intracellular Ca2+ concentration.Ca2+is one of the iost widespread second messengers in eukaryotic cells.Ca2+can activate many different protein kinases and participate in various biological processes such as cell growth,differentiation,movement,and apoptosis.Endoplasmic reticulum(ER)is directly involved in cell damage,leads to the accumulation of unfolded or misfolded proteins in the ER,and disturbances in ER calcium homeostasis.Autophagy is a highly conserved catabolic pathway,which is activated by starvation and stress,and strongly associated with ERS.The accumulation of unfolded or misfolded proteins,and changes in Ca2+concentration induced by ERS can both cause autophagy.Autophagy can in turn inhibit ERS,reduce apoptosis,and promote cell survival.The study aimed to further elucidate the pathogenesis of DGP and explore the effects of IGF-1 on the development of DGP and the underlying mechanism of action,and provide a theoretical basis and experimental evidence for new clinical treatment methods for DGP.Study objective:To observe changes in ERS,autophagy,and PI3K-Akt-PKC pathway-related proteins in gastric smooth muscle tissues of diabetic rats with gastroparesis,investigate the effect of IGF-1 on ERS,autophagy,and PI3K-Akt-PKC-Ca2+ pathway in rat gastric smooth muscle cells cultured under different glucose concentrations,and explore the influence of IGF-1 on development of DGP.Materials and Methods:Diabetic rat model was established,rats were divided into normal control(NC)and 6-week diabetic model(DM6W)groups,changes in the expression of ERS,autophagy-related proteins rat gastric smooth muscle tissues were observed by immunofluorescence staining,the expression of ERS,autophagy and PI3K-Akt-PKC pathway-related proteins was detected by western blot analysis.Rat gastric smooth muscle cells were cultured in vitro for 24 h and 48 h,and were divided into normal glucose 24 h,48 h,high glucose 24 h,48 h,normal glucose+IGF-1 24 h,48 h,and high glucose+IGF-1 24 h,48 h groups.PKC activity in gastric smooth muscle cells was detected by ELISA.Expression of GRP78 a1d LC3 proteins and Ca2+ concentration in cells were observed by confocal laser-scanning microscopy.Changes in the expression of ERS,autophagy and PI3K-Akt-PKC pathway-related proteins in gastric smooth muscle cells were observed by western blot analysis.Results:The diabetic rat model was prepared.The expression of GRP78 and LC3 in gastric smooth muscle tissue between NC group and DM6w group was detected by immunofluorescence staining,the results showed that the expression of GRP78 and LC3 in the DM6w group was significantly increased than that in the NC group.The results of western blot analysis showed that the expression of GRP78,CHOP and LC3II in the DM6w group was significantly higher than that in the NC group(P<0.001).The expression of PI3K,p-Akt,and PKCa was significantly decreased in the DM6w group than that in the NC group(P<0.01).PKC?1 expression was significantly increased in the DM6w group than that in the NC group(P<0.05).Rat gastric smooth muscle cells were cultured in vitro and treated with IGF-1.PKC activity in the gastric smooth muscle cells of each group was analyzed by ELISA,the results showed that at 24 h of culture,no significant difference in PKC activity was found between normal and high glucose groups(P>0.05),after the cells were treated with IGF-1,PKC activity was increased in both normal glucose+IGF-1 and high glucose+IGF-1 groups compared with untreated cells,and the increase in the high glucose+IGF-1 group was more obvious;at 48 h of culture,PKC activity was increased in the high glucose group than that in the normal glucose group(P<0.01);after IGF-1 intervention,there was no significant change in PKC activity between the normal glucose and normal glucose+IGF-1 groups(P>0.05),and PKC activity was significantly increased in the high glucose+IGF-1 group than that in the high glucose group(P<0.05).The changes in intracellular Ca2+ concentration was detected by confocal laser-scanning microscopy,the results showed that after 24 h and 48 h of culture,intracellular Ca2+concentration in cells cultured under high glucose conditions was significantly increased than that in cells cultured under normal glucose condition(P<0.01).After 24 h and 48 h of IGF-1 treatment,intracellular Ca2+concentration was decreased in IGF-1-treated cells cultured under high glucose condition,and was increased in IGF-1-treated cells cultured under normal glucose condition in comparison with the untreated group(P<0.01).Results of confocal laser-scanning microscopy showed that at 24 h of culture,GRP78 expression was significantly increased in the high glucose group compared with the normal glucose group(P<0.05),and there was no significant change in LC3 expression between the two groups(P>0.05);after IGF-1 intervention,GRP78 expression in the normal glucose+IGF-1 group was significantly increased compared with the normal glucose group(P<0.001),there was no significant change between high glucose and high glucose+IGF-1 groups(P>0.05).LC3 expression was decreased in both normal glucose+IGF-1 and high glucose+IGF-1 groups compared with untreated cells(P<0.05).At 48 h of culture,GRP78 expression was significantly increased in the high glucose group compared with the normal glucose group(P<0.001),LC3 expression was decreased in the high glucose group compared with the normal glucose group(P<0.05),after IGF-1 intervention,no significant differences in GRP78 and LC3 expression were found between normal glucose and normal glucose+IGF-1 groups(P>0.05).GRP78 and LC3 expression were both decreased in high glucose+IGF-1 group compared with the high glucose group(P<0.01).Western blot assay results showed that at 24 h of culture,expression of CHOP,GRP78 and LC3? in the high glucose group was increased compared with the normal glucose group(P<0.05).After IGF-1 treatment,CHOP and GRP78 was increased in IGF-1-treated cells cultured under normal glucose condition in comparison with the untreated group(P<0.01),and no significant differences in LC3? expression was found between normal glucose and normal glucose+IGF-1 groups(P>0.05).There was no significant change in the expression of CHOP and GRP78 between high glucose and high glucose+IGF-1 groups(-P>0.05),and LC3? expression was decreased in the high glucose+IGF-1 group than that in the high glucose group(P<0.05).At 48 h of culture,CHOP,GRP78 and LC3? expression were both increased in the high glucose group than that in the normal glucose group(P<0.05).After IGF-1 treatment,there was no significant change in the expression of CHOP and GRP78 between normal glucose and normal glucose+IGF-1 groups(P>0.05),LC3? expression was increased in the normal glucose+IGF-1 group than that in the normal glucose group(P<0.001).The expression of CHOP,GRP78 and LC3? were both decreased in the high glucose+IGF-1 group compared with the high glucose groups(P<0.01).At 24 h of culture,PI3K,p-Akt,PKCa and p-PKCa expression were both decreased in the high glucose group compared with the normal glucose group(P<0.05),PKC?1 and p-PKC?1 expression were increased in the high glucose group compared with the normal glucose group(P<0.05).After IGF-1 treatment,PI3K,PKC?1 and p-PKC?1 expression were both increased,p-PKCa expression was decreased in IGF-1-treated cells cultured under normal glucose condition in comparison with the untreated group(P<0.05),there was no significant difference in p-Akt and PKCa expression between the normal glucose and normal glucose+IGF-1 groups(P>0.05).PI3K,p-Akt,PKCa,p-PKCa and p-PKC?1 expression were increased IGF-1-treated cells cultured high glucose condition in comparison with the untreated group(P<0.05),there was no significant difference in PKC?1 expression between the high glucose and high glucose+IGF-1 groups(P>0.05).At 48 h of culture,PI3K,p-Akt,PKCa,p-PKCa and PKC?1 expression were both decreased in the high glucose group compared with normal glucose group(P<0.05).There was no significant difference in p-PKC?1 expression between high and normal glucose groups(P>0.05).After IGF-1 treatment,PI3K,p-Akt,p-PKC?1 and PKC?1 expression were both increased and PKCa expression was decreased in the normal glucose+IGF-1 group compared with normal glucose group(P<0.05).There was no significant difference in p-PKCa expression between normal glucose and normal glucose+IGF-1 groups(P>0.05).The expression of PI3K,p-Akt,p-PKC?1,p-PKCa and PKC?1 were both increased in high glucose+IGF-1 group compared with high glucose group(P<0.05).There was no significant difference in p-PKCa expression between high glucose and high glucose+IGF-1 groups(P>0.05).Conclusion:Under high glucose condition,IGF-1 can improve disturbance of intracellular Ca2+ homeostasis,inhibit the development of ERS and autophagy,exert protective effects on rat gastric smooth muscle cells by activating the PI3K-Akt-PKC pathway in cells. |
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