Jianpi Yifei ? Reduces Apoptosis Of COPD Airway Epithelial Cells By Regulating ROS-ER Stress-Ca²⁺ Signaling Pathway

BackgroundSmoking is recognized as the most dangerous cause of chronic obstructive pulmonary disease(COPD).Reactive oxygen species(ROS)in cigarette smoke can cause oxidative stress and lead to the development of COPD.ROS can induce apoptosis of lung airway epithelial cells,but the mechanism of action is not yet clear.This study intends to use in vitro and in vivo experimental models to evaluate the protective effects of Jianpi Yifei ?(JPYF ?)on airway epithelial cells stimulated by CSE and airway epithelial cells in COPD mice,with a focus on JPYF ? by regulating the ROS-ER stress-Ca²⁺signal pathway,it can reduce the apoptosis mechanism of COPD airway epithelial cells,and provide scientific basis for JPYF ? clinical treatment of COPD.Methods1.In vivo experiments,60 BALB/c mice were randomly divided into 5 groups:Control group,CS group,CS+JPYF ? low-dose group,CS+JPYF ?middle-dose group and CS+JPYF ? high-dose group.The model of COPD was established by cigarette smoke fumigation.The control group did not do any treatment,While the CS group smoked cigarette for one month.The JPYF ?group,the different dose of JPYF ? was administered to the stomach once a day during the last two weeks of cigarette smoke model,and the mental state,activity,diet,and fur condition of the mice were recorded during the experiment;Mice lung tissue,TUNEL test was used to detect the protective effect of JPYF ? on the epithelial cells in the trachea of COPD mice;immunohistochemistry was used to detect the effects of JPYF ? on the trachea of COPD mice regulation of ER stress-related proteins(GRP78,p-e1F2 a and CHOP).2.Cigarette smoke extract(CSE)was used in vitro to stimulate human lung airway epithelial cells(Beas-2B and 16-HBE)to construct a COPD model.Use MTT method to detect the toxicity of JPYF ? on airway epithelial cells;Use MTT method to detect the protective effect of JPYF ? on airway epithelial cells stimulated by CSE.Flow cytometry was used to detect the protective effect of JPYF ? on airway epithelial cells stimulated by CSE;Hoechest 33342 staining was used to detect the protective effect of JPYF ? on airway epithelial cells stimulated by CSE Effect;Flow cytometry was used to detect the effects of JPYF ? on the cycle of airway epithelial cells stimulated by CSE;Western blot was used to study the regulating effect of JPYF ? on apoptosis-related proteins(Caspase-3,Cleaved-caspase-3,Bax and Bcl-2)and ER stress-related proteins(GRP78,PERK,p-PERK,eIF2 ?,p-eIF2 ? and CHOP)in airway epithelial cells stimulated by CSE.Study the correlation between JPYF ? inhibit ROS production in airway epithelial cells and its inhibition of ER stress and reduce apoptosis(including the detection of ROS in JPYF? on CSE-stimulated airway epithelial cells by flow cytometry Regulatory effect of production;Western blot to study the correlation of JPYF ? to reduce ROS production in CSE-stimulated airway epithelial cells and its inhibition of ER stress;MTT to detect JPYF ? to reduce CSE-stimulated airway epithelium Correlation between ROS production in cells and its inhibition of apoptosis);Study the JPYF ? correlation between the reduction of Ca²⁺concentration in airway epithelial cells and its inhibition of ER stress and apoptosis(including the use of flow cytometry to detect the regulatory effect of JPYF ? on reducing the concentration of Ca²⁺ in airway epithelial cells stimulated by CSE;Western blot was used to study the correlation between JPYF ? reducing Ca²⁺ concentration in airway epithelial cells stimulated by CSE and its inhibition of ER stress;MTT was used to detect the correlation between JPYF ? reducing Ca²⁺ concentration in airway epithelial cells stimulated by CSE and its inhibition of apoptosis).Results1)General condition of the mice:The mice in the Control group had good mental state,uniform breathing,sensitive response,shiny and smooth hair color,and normal diet.The mice in the CS group suffered from poor mental state,shortness of breath,slow response,poor diet,and messy hair color.When the JPYF ? group started to administer in the last two weeks,the degree of shortness of breath,response sensitivity,diet,hair color and other aspects were improved compared with the CS group.2)JPYF ? can improve the severity of apoptosis of lung airway tissue in COPD mice(p<0.05).3)JPYF ? can reduce the expression of ER stress-related proteins(GRP78,p-elF2 ? and CHOP)in the trachea of lung tissue of COPD mice(p<0.05).4)JPYF ? has no toxicity to airway epithelial cells(p>0.05),and can reduce the toxicity of CSE to cells(p<0.05).5)CSE can inhibit the activity of Beas-2B and 16-HBE cells in a time-and concentration-dependent manner and induce apoptosis.6)Compared with normal airway epithelial cells,the number of apoptosis in airway epithelial cells stimulated by CSE increased significantly(p<0.05).However,JPYF ? can reduce the apoptosis of airway epithelial cells induced by CSE(p<0.05).7)Compared with normal airway epithelial cells,the cell cycle of airway epithelial cells stimulated by CSE is blocked in G0/G1 phase.However,JPYF? can reduce the blockade of CSE-induced airway epithelial cells in the G0/G1 phase(p<0.05).8)JPYF ? can reduce the pro-apoptotic protein expression including Cleaved-caspase-3 and Bax,increase the expression of anti-apoptotic protein Bcl-2(p<0.05),and reduce the expression of ER stress related proteins GRP78,p-PERK,p-eIF2 ? and CHOP(p<0.05).9)JPYF ? and the antioxidant NAC(5 mM)can inhibit the production of ROS and the expression of GRP78,p-PERK,p-eIF2 ? and CHOP in airway epithelial cells induced by CSE(p<0.05).JPYF ? and antioxidant NAC can increase the vitality of airway epithelial cells induced by CSE,and the effect is best in the JPYF ?+NAC group(p<0.05).10)JPYF ? and endoplasmic reticulum inhibitor TUDCA(1 mM)can inhibit the production of ROS induced by CSE in airway epithelial cells and ER stress-related proteins GRP78,p-PERK,p-eIF2? and CHOP expression(p<0.05).JPYF ? and endoplasmic reticulum inhibitor TUDCA can increase the viability of airway epithelial cells induced by CSE,and the effect is best in the JPYF ?+TUDCA group(p<0.05).11)JPYF ? and Ca²⁺ inhibitor BAPTA-AM(10 ? M)can inhibit the production of ROS induced by CSE in airway epithelial cells and ER stress-related proteins GRP78,p-PERK,p-eIF2 a and CHOP expression(p<0.05).JPYF ? and calcium inhibitor BAPTA-AM can increase the viability of airway epithelial cells induced by CSE.The effect is best in the JPYF ?+BAPTA-AM group(p<0.05).12)JPYF ? and IP3R inhibitor 2-APB(50 ? M)can inhibit the production of ROS induced by CSE in airway epithelial cells and the expression related to ER stress proteins including GRP78,p-PERK,p-eIF2 ? and CHOP(p<0.05).JPYF ? and IP3R inhibitor 2-APB can increase the viability of airway epithelial cells induced by CSE,and the effect is best in the JPYF ?+2-APB group(p<0.05).Conclusion1.JPYF ? has a therapeutic effect on CS-induced COPD mice by improving the apoptotic expression of lung tissue in mice and repairing damaged airways and their tissues.2.JPYF ? can protect the apoptosis of Beas-2B and 16-HBE cells stimulated by CSE.The anti-apoptotic effect may be related to the interruption of ROS-ER stress-Ca²⁺ signaling pathway.