Study On The Role And Mechanism Of Paxillin In HPS-related Abnormal Proliferation Of PASMCs

Background and objective:Hepatopulmonary syndrome (HPS) occurs mainly in patients with end-stage liverdisease including cirrhosis. The incidence of HPS is more than15%, while the mortalityrate of which increased year by year. Therefore, HPS become a hot topic gradually.Liver-derived cytokines leading to intraplmonary vasodilatation (IPVD) which is the reasonof PASMCs abnormal proliferation. The PASMCs proliferation further aggravate pulmonaryvascular remodelling (PVR) ultimately. Therefore, the main pathological features ofHPS-related PVR is the abnormal proliferation of PASMCs. Our previous study found that,the HPS rat serum and hypoxia induced abnormal proliferation of PASMCs; meanwhile,phenotype modulation of PASMCs and diversification of the cytoskeletal proteinsexpression have taken place. Previous studies have found that in some diseases (such aspulmonary hypertension) pathological process, PASMCs abnormal proliferation, whileaccompanied PASMCs phenotype modulation and cytoskeletal rearrangement. Maybe, thePASMCs phenotype modulation and cytoskeletal rearrangement are the upstream pathwaysof their abnormal proliferation. To find the relationship between cytoskeletal proteins andcell proliferation, which is conducive to further explain the molecular mechanisms of HPS.Paxillin is a phosphorylated protein. Previous studies have found that it expressed invarious cells and played important roles in a variety of cell biological function. Paxillin actimportant regulatory role in the expression and rearrangement of cytoskeletal proteins.Previous studies have found that annexin A1(ANXA1) and paxillin may interact with eachother. ANXA1may affect paxillin expression. So, research on paxillin is a preliminarystudy on the deep and has the vital significance. Based on the above evidences, wehypothesized that: in the molecular mechanisms on HPS-related abnormal PASMCsproliferation, the changes of cytoskeletal proteins expression induced cytoskeletalrearrangement and PASMCs phenotype modulation, which resulting in abnormal proliferation of PASMCs; paxillin may play a key regulatory role in regulating thecytoskeletal proteins expression, and affect the proliferation of PASMCs ultimately. Thisstudy intends to build a rat model of HPS by common bile duct ligation (CBDL), primaryculture PASMCs, detect the proliferation of PASMCs by CCK-8and3H-TdR, detectpaxillin and cytoskeletal proteins expression by RT-PCR and Western-blot; against paxillinexpression by siRNA intervention. Through the above technology to explore the molecularmechanisms of paxillin in HPS-related abnormal PASMCs proliferation. And to providenew measures for the prevention and treatment of HPS.Methods:Total two parts:(1) Changes of the expression of Paxillin and cytoskeletal proteins in PASMCs inducedby HPS rat serum.1) To build HPS model of rat and prepare HPS rat serum.2) The isolation, cultivation and identification of PASMCs of rat.3) RT-PCR and Western-blot were used to assay the expression of paxillin.4) Western-blot is used to assay cytoskeletal proteins (α-actin, α-tubulin, destrin)expression.(2) The influence of changing paxillin expression on the proliferation and cytoskeletalproteins expression of PASMCs which were cultured by HPS rat serum.1) siRNA reduced the expression of paxillin in PASMCs.2) The influence of down-regulated paxillin expression on the proliferation andcytoskeletal proteins expression of PASMCs which were cultured by HPS rat serum.Results:(1) HPS rat serum up-regulated paxillin transcription level and protein expressionsignificantly. The transcription level and protein expression increased in association withtreatment time.(2) HPS rat serum down-regulated the expression of cytoskeletal protein (destrin, α-actin, α-tubulin)in PASMCs in association with treatment time.(3) Paxillin protein expression was inhibited by specific paxillin siRNA effectively.(4) Down-regulation of paxillin by siRNA results in the inhibition of the dysregulationof cytoskeletal proteins and proliferation of PASMCs. Conclusion:The above results are summarized, on the abnormal proliferation of PASMCs associatewith HPS, changes in expression of cytoskeletal proteins may be the premise of theabnormal proliferation; and in the process, paxillin may act as a key regulator of target,which can regulate PASMCs proliferation by changing the expression of cytoskeletalproteins, namely: paxillin play an important role in the regulation of PASMCs proliferationralated HPS.