Pretreatment With Perphenazine Alleviated Endoplasmic Reticulum Stress Induced By Tunicamycin

Objective : The endoplasmic reticulum is an important organelle that is mainly responsible for membrane protein,secreted protein,and lipid synthesis,modification,and processing.When misfolded proteins accumulate in the endoplasmic reticulum,it activates endoplasmic reticulum stress,leading to unfolded protein response(UPR),which induces the expression of chaperone and proteins involved in the recovery process.Induction of ER stress may be protective,but it is cytotoxic and leads to cell death if overactivation continues.Endoplasmic reticulum stress is involved in the pathogenesis of many diseases,such as pulmonary fibrosis,bronchopulmonary dysplasia(BPD),and lung cancer.Multiple evidences suggest that endoplasmic reticulum stress-induced cell dysfunction and cell death are the major causative factors of many diseases.Therefore,regulatory factors involved in endoplasmic reticulum stress may become potential targets for therapeutic discovery.There are two ways to reduce ER stress: ER-associated protein degradation(ERAD)pathway and ER-phagy pathway.A common definition of ERAD is that defective proteins are eventually expelled from the endoplasmic reticulum and degraded in the cytoplasm by the ubiquitin proteasome system,which degrades soluble proteins in UPR.ER-phagy is a kind of selective autophagy,which double membranes in autophagosomes are at least partially derived from the endoplasmic reticulum and degrade insoluble proteins in the endoplasmic reticulum.ER-phagy is a constitutive or regulated fragmentation that transports endoplasmic reticulum fragments to the lysosome to clear it,and plays an important role in maintaining intracellular homeostasis,fat balance,and protein balance.Cell-cycle progression gene 1(CCPG1)is a resident protein in the endoplasmic reticulum,which specifically binds microtubule associated protein 1 light chain 3(LC3)and FIP200 proteins.These interactions promote endoplasmic reticulum autophagy.UPR can induce CCPG1,thus linking endoplasmic reticulum stress with endoplasmic reticulum autophagy and driving endoplasmic reticulum degradation.The MLE12 cell line is an approximate cell line of mouse type II alveolar epithelial cells,and the lysosome-derived organelle lamellar bodies are the hallmark organelles of type II alveolar epithelial cells.We observed that perphenazine pretreatment interfereswith the lysosomal function of endosome and enhances the ability of cells to resist endoplasmic reticulum stress induced by tunicamycin,to analyze the mechanism of perphenazine pretreatment to relieve endoplasmic reticulum stress and its effect on differentiation of MLE12 cells.Methods: 1.Western blot,Real-time PCR and PCR techniques were used to detect the expression levels of Bip protein,mRNA and s-XBP1/u-XBP1 in MLE12 cells pretreated with perphenazine at different concentrations for 24 h respectively,and then endoplasmic reticulum stress was induced by tunicamycin.2.In order to detect the effect of perphenazine and tunicamycin on MLE12 cells on endoplasmic reticulum stress,we used western blot and Hoechst33342 staining to detect the corresponding Bip protein and cell health status after perphenazine pretreatment and non-pretreatment.3.In order to further explore the effect of perphenazine on MLE12 cells autophagy and cholesterol in lysosomes,we used western blot to detect the expression levels of LC3 II and P62 proteins after different concentrations of perphenazine treatment for 24 h and the same concentration for different times.Filipin staining was used to detect the accumulation of cholesterol in lysosomes after treatment with different concentrations of perphenazine for 24 h and at the same concentration for different times.4.In order to further study whether perphenazine pretreatment relieves endoplasmic reticulum stress induced by tunicamycin is related to autophagy,we used western blot to detect the expression level of Bip protein after perphenazine treatment for 3h and 10 h and then tunicamycin induced endoplasmic reticulum stress.we used western blot to detect the expression level of CCPG1 protein after pretreatment of MLE12 cells with perphenazine at different concentrations for 24 h,and then induced endoplasmic reticulum stress with tunicamycin.Hoechst33342 staining was used to detect nuclear shrinkage after pretreatment of 3?M perphenazine for 24 h,endoplasmic reticulum stress induced by tunicamycin and addition of lysosomal inhibitor Bafilomycin and 26 S proteasome inhibitor(MG132).5.To investigate the effect of perphenazine treatment on the differentiation of MLE12 cells,we used Real-time PCR to detect the expression levels of SPB and SPC mRNA of MLE12 cells treated with 3?M perphenazine 6h,12 h,24h,and 48 h.Results: 1.Western blot and Real-time PCR results showed that compared with untreated cells,pretreatment with 3?M perphenazine could alleviate endoplasmic reticulum stress induced by tunicamycin,and the expression of Bip protein and mRNA decreased,the ratio of s-XBP1 / u-XBP1 decreased and apoptotic cells decreased.2.The results of western blot and Hoechst33342 showed that the simultaneous treatment of perphenazine and tunicamycin could not alleviate endoplasmic reticulum stress and increase apoptotic cells.3.Western blot and Filipin staining showed that LC3 II and P62 proteins increased,autophagy level increased and cholesterol in lysosomes significantly aggregated after 8h of 3?M perphenazine treatment compared with the control group.This indicates that low dose perphenazine can enhance autophagy initiation of MLE12 cells and affect the function of endosome lysosomes.4.Western blot results showed that perphenazine pretreatment for 10 h could relieve endoplasmic reticulum stress,but there was no significant change in pretreatment for 3h.Western blot analysis showed that the expression of CCPG1 protein increased after 24 h of treatment of MLE12 cells with perphenazine at 3?M.Hoechst33342 staining results showed that a large number of cells died after treatment with Bafilomycin inhibitor,while MG132 inhibitor had no significant change.The above results indicate that the remission of endoplasmic reticulum stress by perphenazine pretreatment may be related to the endoplasmic reticulum autophagy pathway.5.Real-time PCR results showed that the mRNA expression level of differentiation marker SPB increased after perphenazine pretreatment of MLE12 cells for 12 h and SPC mRNA expression level increased after 48 h.The results showed that perphenazine treatment promoted differentiation into type II alveolar epithelial cells.Conclusion: Low-dose perphenazine pretreatment enhances the autophagy level of MLE12 cells,enhances the ability of the cells to resist tunicamycin-induced endoplasmic reticulum stress,and promotes their differentiation into type II alveolar epithelial cells.