| Objective: To investigate the effects of saponin on endoplasmic reticulum stressors GRP78 and ATF6 in rat gastric interstitial cells of Cajal induced by tunicamycin.Methods:(1)The primary rat gastric interstitial cells of Cajal(ICCs)were isolated by enzymatic hydrolysis,and the c-kit antibody of ICCs was identified by immunofluorescence staining;the different concentrations were detected by CCK-8 method(0?g/ml,0.125?g/ml,0.25?g/ml,0.5?g/ml,1?g/ml,2?g/ml)The effect of Tm on the activity of ICCs at different time(12h,24 h,48h);expression of GRP78 mRNA in ICCs that the key molecule of endoplasmic reticulum stress by qRT-PCR;the ultrastructure of the endoplasmic reticulum of ICCs was observed by electron microscopy.(2)Effect of zhishi on endoplasmic reticulum stress ICCs: Serum pharmacology was used to prepare zhishi-serum,and inhibitor 4-PBA blocked endoplasmic reticulum stress.ICCs were randomly divided into normal group(normal ICCs culture),Tm group(1 ?g/ml Tm treated ICCs),inhibitor group(1 ?g/ml TM + 10 mM 4-PBA combined)and zhsihi group(1 ?g/ml Tm)+ 20% combined with drug-serum),each group of cells were used for detection for 24h: the ultrastructure of endoplasmic reticulum of each group of ICCs was observed by transmission electron microscopy;the dehydrogenation of lactate in each group of ICCs was determined by microplate.LDH activity in each group of ICCs were determined by Microplate method;ROS level in each group of ICCs was determined by fluorescence microplate reader;ICCs activity of ICCs in each group was detected by CCK-8 method;migration and repair ability of ICCs in each group was observed by cell scratch test;The expression of GRP78 and ATF6 in ICCs were detected by Elisa,qRT-PCR,Western blot.Results:(1)The cells are fusiform,triangular,star-shaped,accompanied by slender branching,less cytoplasm,large nuclei,and synaptic links with surrounding cells under the microscope.Immunofluorescence staining shows c-kit was positively expressed;the effect of Tm on ICCs activity was detected by CCK-8: when Tm was 2?g/ml,ICCs were cultured at 12 h,24h and 48 h,the ICCs activity was decreased,12h(OD)was 0.553±0.026(P<0.05),24h(OD)was 0.466±0.046(P<0.05),48h(OD)was 0.164±0.034(P<0.05).When Tm was 1 ?g/ml,ICCs were cultured at 24 h and 48 h,the ICCs activity were all decreased,24h(OD)was 0.736±0.008(P<0.05)and 48h(OD)was 0.359±0.079(P<0.05).The qRT-RCR results showed that compared with 0?g/ml Tm,the expression of GRP78 mRNA of 1?g/ml Tm were cultured after 24 hours of ICCs were increased(P<0.05).The results of transmission electron microscopy showed that 0?g/ml Tm acted on ICCs for 24 h,and the endoplasmic reticulum structure were complete and neatly arranged;after 1?g/ml Tm acts on ICCs for 24 h,the cell endoplasmic reticulum structure is swollen,expanded,vesiculation,degranulation change.(2)Effect of zhishi on endoplasmic reticulum stress of ICCs: Transmission electron microscopy showed that the endoplasmic reticulum of ICCs in normal group was normal,and the endoplasmic reticulum of ICCs in Tm group was swollen,dilated,vesicular,and degranulated.The endoplasmic reticulum of ICCs in the inhibitor group was ruptured and vesicularized.The endoplasmic reticulum of ICCs in the zhishi group was swollen and showed localized expansion.The LDH activity of each group of ICCs was compared: compared with the normal group,the LDH activity of the Tm group,the inhibitor group,and the zhishi group were all increased(P<0.05).Compared with the Tm group,the LDH activity of the inhibitor group and the zhishi group were decreased(P<0.05).The ROS of ICCs each group was compared : compared with the normal group,the ROS of the Tm group,the inhibitor group,and the zhishi group were all increased(P<0.05);compared with the Tm group,the ROS of ICCs in the inhibitor group and the zhishi group decreased(P<0.05).The fluorescence intensity in the cells was strong under the inverted fluorescence microscope in the Tm group;while the fluorescence intensity in the cells was decreased after treatment with the inhibitor and zhishi-serum.The percentage of ICCs survival was compared: compared with the normal group,the percentage of ICCs survival of Tm group,inhibitor group,zhishi group were decreased(P<0.05);compared with the Tm model group,the percentage of ICCs survival of ICCs in zhishi group and the inhibitor group were all increased(P<0.05).The results of ICCs scratch test showed that the cell spacing in the same time period zoom out over time.After 12 hours of were cultured,the scratch healing rate of the normal group was(38.0±2.0)%,the scratch healing rate of the Tm group was(19.7±4.5)%,and the scratch healing rate of the inhibitor group was(26.3±3.7)%,the scratch healing rate of the cells in the tamping group was(30.0±2.0)%;after 24 hours of were cultured,the scratch healing rate of the normal group was(31.0±2.0)%,and the cell scratch healing rate of the Tm group was(7.0±3.6),the scratch healing rate of cells in the inhibitor group was(35.7±2.1)%,and that in zhishi group was(38.3±3.1)%.Compared with the Tm group,the scratch healing rate of the ICCs in the tamping group was high(P<0.05);Elisa assay showed that compared with the normal group,the GRP78 and ATF6 protein contents in the Tm model group,the inhibitor group and zhishi group were increased(P<0.05);compared with the Tm group,the GRP78 and ATF6 protein contents in the inhibitor group and the phlegm group were decreased(P<0.05).Compared with the normal group,the expressions of GRP78 and ATF6 mRNA in the Tm model group,the inhibitor group and zhishi group were increased(P<0.05).Compared with the Tm group,the expression of GRP78 and ATF6 mRNA in the sputum group decreased.(P<0.05);Compared with the inhibitor group,the expression of GRP78 mRNA in the inhibitor group and zhishi group were decreased(P<0.05);compared with the normal group,the protein expressions in ICCs of GRP78 and ATF6 of in the Tm model group,the inhibitor group and zhishi group were increased(P<0.05).Compared with the Tm group,the protein expressions in ICCs GRP78 and ATF6 in the inhibitor group and zhishi group were decreased(P<0.05).Conclusion:The model of endoplasmic reticulum stress in rat gastric ICCs was successfully constructed using tunicamycin;sputum can alleviate the endoplasmic reticulum stress damage induced by tunicamycin,improve the growth activity of damaged cells,and improve the cell repair ability.It may be related to its inhibition of endoplasmic reticulum stress ATF6 pathway. |
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