KLOTHO Expression In Human Bronchial Epithelial Cells:Effects On Inflammation And Oxidant Injury

Objective: Human Klotho(KL)is an anti-ageing protein that protects cells against inflammation and damage.We previously demonstrated that KL was mainly distributed along the airway epithelium and its expression was decreased in the lungs of smokers and further reduced in patients with COPD.In this study,we investigated the mechanisms that control KL expression and function in the airways.Methods: The effect of cigarette smoke extract(CSE)and tumor necrosis factor alpha(TNF-α)on KL expression and secretion was detected in cultured human bronchial epithelial cells(16HBE)using RT-PCR,Western blotting(WB)and enzyme-linked immunosorbent assay(ELISA)methods.Moreover,the effect of KL on CSE-mediated inflammation and hydrogen peroxide(H2O2)-induced cellular injury/apoptosis was determined using small interfering RNAs.RT-PCR and ELISA were used respectively to detect the transcription and secretion level of the cytokines(IL-8,IL-6,MCP-1),and flow cytometry was used to measure the cellular oxidative stress injury and apoptosis ratio.Meanwhile,immunofluorescence as well as WB methods were adopted to explore KL-related pathways.Results: We found that CSE and TNF-α decreased KL expression and release from airway epithelial cells,which was associated with enhanced release of proinflammatory cytokines(P<0.05),and the NF-κB pathway inhibitors(JSH23)significantly reversed this effect of TNF-α(P<0.05).Moreover,KL depletion by using KL-specific si RNAs showed decreased cell viability,increased apoptosis level and elevated cytokines(IL-8,IL-6 and MCP-1)m RNA expression(P<0.05).We also found that KL deletion increased cigarette smoke-induced inflammatory factor(IL-8)expression and secretion,and exacerbated the activation of related inflammatory pathways(mainly NF-κB and ERK1/2 pathway)(P<0.05).In addition,KL depletion aggravated cell damage induced by H2O2 measured by further ROS production,reduced cell viability,and enhanced apoptosis(P<0.05).These may relate to the phosphorylation of protein kinase B(Akt/PKB),extracellular regulated protein kinase(ERK1/2)and p38 MAPK pathways and excessive consumption of nuclear transcription factor NRF2,which is an important intracellular antioxidant(P<0.05).Conclusions: Reduced KL expression in COPD airway epithelium was associated with increased oxidative stress,inflammation,and apoptosis.These findings of KL functions in human airway epithelial cells not only provide valuable supplement to previous studies in this regard,but also establish its exact role in the pathogenesis of COPD and may open up new perspectives to intercept pathways responsible for accelerated ageing and restore the natural history of senescence within the COPD lung.