Capsaicin Inhibits The Apoptosis Of Rat Alveolar Epithelial Type Ⅱ Cells Undergoing Hypoxia/Reoxygenation

Purpose: Our experiment aimed to explore whether capsaicin would affect ratalveolar epithelial type Ⅱ cells (AT Ⅱ) apoptosis after they underwenthypoxia/reoxygenation (HR) and the possible pathways of cell death in vitro.Method: ATⅡwere inoculated at the density of0.7~0.8×10^5/ml in six well plate,cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with10%fetal bovine serum (FBS) in an atmosphere of37℃5%CO2incubator, and identifiedby transmission electron microscope and immunocytochemistry (SABC-DAB). ATⅡwhich had been inoculated and identified successfully were divided into six groups:control group (group C), hypoxia/reoxygenation group (group HR), capsaicin wasadded1h before hypoxia group (group CapB), capsaicin was added at the beginningof hypoxia group (group CapH), capsaicin was added at the beginning ofreoxygenation group (group CapR) and capsazepine group (group CapZ). All groupswere duplicated10times except group CapZ was duplicated3times. The cells werenormal without any treatment in group C. The cells were used Hanks’ solutionwithout glucose to24h hypoxia in an atmosphere of37℃1%O294%N25%CO2inanoxic incubator and used DMEM without FBS to24h reoxygenation in an aerobicincubator in group HR. The cells were respectively pre-treated with50uM capsaicin1h before hypoxia in group CapB, dealt with50uM capsaicin at the beginning ofhypoxia in group CapH and50uM capsaicin at the beginning of reoxygenation after24h hypoxia in group CapR, other treatments as group HR. The cells werepre-conditioned with10uM capsizepine1.5h before hypoxia and dealt as same asgroup CapH in group CapZ. The alterations of morphology of ATⅡwere overviewedby inverted microscope and the apoptosis was tested by flow cytometry after HR.Lastly, in order to detect the expression of mRNA of caspase-9and confer thepossible mechanisms of ATⅡ apoptosis, group C cells growing70-80%confluence,group HR and group CapH undergoing24h hypoxia and24h reoxygenation were examined by realtime fluorescence quantitative polymerase chain reaction(Q-PCR)(n=6).Results:(1) ATⅡcontains characterized lamellar body in the cytoplasm, expressespulmonary surfactant-associated protein A(SP-A) and C(SP-C).(2)①HR induced rat ATⅡ apoptosis and its morphology altered. Cells attachedto plates and connected to each other, plasma membrane and cell nucleus all keptintact, supernatant liquid was clean in group C. After HR, cells attached to platesdecreased obviously in group HR, group CapB, group CapH, group CapR and groupCapZ, cells turned round, some became granular, the membrane was intact butnucleus was broken, white granular cells floated on supernatant.②Compared withgroup HR, much more cells attached to plate and there were less supernatant cells ingroup CapH; but there’s no significantly difference in group CapB, group CapR andgroup CapZ;③Compared with group CapH, supernatant cells increased in groupCapZ.(3) Flow cytometry analysis reveals that:①Compared with group C, theapoptosis rate of rat ATⅡ in group HR, group CapB, group CapH, group CapR andgroup CapZ apparently increased (p<0.05).②Compared with group HR, theapoptosis rate evidentedly decreased in group CapH (p<0.05); while the apoptotic rateof ATⅡnot obviously altered in group CapB, group CapR and group CapZ(p>0.05).③In contrast to group CapB and group CapR, the apoptotisis rate dropped apparentlyin group CapH (p<0.05).④Compared with group CapH, the apoptotic rate of ATⅡingroup CapZ manifestly increased(p<0.05).(4)Compared with group C, the level of mRNA of caspase-9increased nearly3fold in group HR; but slightly increased in group CapH. Compared with group HR,the level of mRNA of caspase-9diminished nearly2fold in group CapH.Conclusion:(1)Capsaicin has protective effect on rat ATⅡ cells apoptosis inducedby hypoxia/reoxygenation, especially added capsaicin at the beginning of hypoxia,which it can lower their apoptosis.(2)Capsaicin may serve its role of protection viaTRPV1receptor.(3)Apoptosis of ATⅡ cells caused by HR perhaps correlates tostimulate caspase-9in the intrinsic pathway. Capsaicin reduces the cell death possibly through decreasing the level of caspase-9mRNA.