Bufei Yishen Recipe Regulates The Influence Of Klotho Gene On The Intervention Of RIG-I/NF-?B Pathway In BEAS-2B Cell Line By Cigarette Extract

Part 1 Effects of Klotho gene expression on inflammatory response and RIG-I / NF-?B pathway in CSE-treated BEAS-2B cell linesObject:Chronic obstructive pulmonary disease is a disease characterized by persistent airflow limitation,which can be prevented and treated.Airway limitation is progressive development,and its chronic inflammatory response is enhanced with airway and lung tissue sensitive to harmful gases such as tobacco smoke and harmful particles.Long-term exposure to smoke can induce oxidative stress in the lungs,causing cell senescence or aging.Although Klotho as anti-aging protein,more and more studies have shown Klotho protein has anti-inflammatory effect.Studies have shown that in the aging rat kidney,Klotho protein as an inhibitor of intracellular inflammatory response to down-regulation of retinoic-acid-inducible gene-I(RIG-I)/NF-?B signaling pathway to play anti-inflammatory anti-aging effect.The intracellular klotho protein inhibits the expression of IL-6 by inhibiting RIG-I.We hypothesized that Klotho protein plays a role in down-regulating IL-6 expression by mediating RIG-I / NF-?B pathway in COPD.In this study,cigarette smoke extract(CSE)was diluted in BEAS-2B cell culture medium to prepare COPD cell model.Construction of klotho protein by expressionand overexpression of the plasmid,transfected into CSE intervention cells.The expression of Klotho,RIG-I,NF-?B,IL-6 and TNF? were determined to determine the anti-inflammatory effect of klotho gene and its relationship with RIG-I / NF-?B pathway.Method:1.Cell culture and CSE intervention The BEAS-2B human lung epithelial cell line was taken from the American Standard Culture Collection and the cigarette extract was diluted with cell culture prior to use.2.Preparation of Klotho overexpressing cells Construction of klotho overexpression plasmids to elucidate the overexpression of BEAS-2B cells with stable expression of klotho.3.The mRNA expression of Klotho,NF-kB / p65 and RIG-I was detected by qRT-PCR.4.The expression of Klotho,NF-kB / p65 and RIG-I protein was detected by Western blotting.5.The expression of IL-6 and TNF? was detected by ELISA.Results:1.The expression of klotho gene was decreased in CSE-induced lung epithelial cell lines.In contrast,the expression of RIG-I,NF-?B,IL-6 and TNF-? increased2.In the CSE intervention cell line,the concentrations of IL-6,TNF?,RIG-I and NF-?B in the Klotho overexpression group were lower than those in the Klotho normal group.Conclusion:The expression of klotho protein was decreased in the CSE-treated COPD model.Overexpression of klotho protein inhibits the effects of IL-6,TNF?,RIG-I,NF-?B.Indicating that Klotho protein has anti-inflammatory effect in COPD model cell lines and may play a role in down-regulating the expression of IL-6 and TNF? by mediating RIG / NF-?B pathway.Part 2 Effect of Bufei Yishen Formula on Klotho Gene Expression and Inflammatory Related RIG-I / NF-?B Pathway in CSE-treated BEAS-2B cell linesObject:The goal of treatment for chronic obstructive pulmonary disease is to relieve symptoms,improve endurance and health status,prevent disease progression,prevent acute exacerbations,and ultimately reduce mortality.The current treatment(including bronchodilators,glucocorticoids)is mostly to improve the symptoms and anti-inflammatory,it is difficult to fight against the process of lung aging.Chinese medicine theory: in terms of COPD,by adjusting the lung and kidney,can gradually change the patient's body environment,enhance its physical,and thus achieve the purpose of reducing recurrence.This highlights the use of the overall concept of traditional Chinese medicine treatment of the unique advantages.Our previous study found that the use of Bufei Yishen Paste for the treatment of stable COPD,it can improve the clinical symptoms and lung function;The method used in clinical safety,and has a definite effect.Bufei Yishen Formula composed of six herbs,namely Astragalus,Ginseng,Rehmannia,cornus,Xianling spleen and Atractylodes.Six kinds of traditional Chinese medicine have anti-inflammatory and anti-aging effect.Klotho protein as an anti-aging protein,it not only has anti-aging effect,but also has anti-inflammatory effect.Increased expression of Klotho protein can inhibit the inflammatory response caused by COPD.We speculated that Bufei Yishen Formula can increase the expression of Klotho gene,mediate RIG-I / NF-?B pathway,inhibit the inflammatory response,so as to achieve the purpose of treatment of COPD.In this study,we proposed the intervention of Bushenyiqi formula and dexamethasone in COPD cell model.The expression of Klotho,RIG-I and NF-?B was detected by RT-PCR and Western blot.The expression of IL-6 and TNF? was detected by ELISA.Method:1.Bufei Yishen traditional Chinese medicine compound cytotoxicity determination: When BEAS-2B cells all adherent growth,determination of Bufei Yishen Chinese medicine compound maximum non-toxic dose.2.Compared with dexamethasone and blank control group,the expression of Klotho,NF-k B / p65 and RIG-I m RNA and protein were detected by q RT-PCR and Western blotting.3.The expression of IL-6 and TNF? was detected by ELISA before and after the intervention,compared with the dexamethasone group and the blank control group.4.In the CSE intervention,when using Si RNA technology knock low klotho expression,we explore whether the Bufei Yishen Formula can still play an anti-inflammatory effect.Results1.The expression of klotho gene was increased and the expression of RIG-I,NF-?B,IL-6 and TNF-? were decreased after the addition of Bufei Yishen Formula to CSE-treated lung epithelial cell line.2.Bufei Yishen Formula can inhibit the inflammatory response.It can inhibit the expression of IL-6,TNF?,RIG-I and NF-?B by up-regulating the expression level of klotho.But its effect is weaker than dexamethasone.3.Under CSE intervention,when knocking low klotho expression,Bufei Yishen Formula anti-inflammatory effect was significantly reduced.Conclusion:Bufei Yishen Formula can promote the expression of klotho gene and inhibit the expression of IL-6,TNF?,RIG-I and NF-?B,indicating that Bufei Yishenfang Formula has definite anti-inflammatory effect.The anti-inflammatory effect was significantly decreased after knocking low klotho.It is suggested that the anti-inflammatory effect of Bufei Yishen Formula may be the up-regulation of klotho protein,which can play a role in mediating the expression of IL-6 and TNF? by mediating RIG-I / NF-?B pathway.