Hypoxia-precondition Enhance The Therapeutic Effects Of Mesenchymal Stem Cell In Hepatic Ischemia Reperfusion Injury

Objectives1.Isolating and identifying rat bone marrow-derived mesenchymal stem cells(BMMSC),compare proliferation,homing ability among conventional cultured BMMSC(n-BMMSC),hypoxia-precondition BMMSC(n-BMMSC).2.Compare the therapeutic effects among n-BMMSC,hp-BMMSC,rapa-hp-BMMSC on hepatic ischemia reperfusion injury(HIRI)model rats and the variation trend of these effects with time.3.Study mechanisms of different therapeutic effects of n-BMMSC,n-BMMSC,rapa-hp-BMMSC on HIRI and provide mechanisms reference for enhancing the efficacy of BMMSC-based therapies on HIRI.Methods1.BMMSC were isolated from SD rats at the of age 5 weeks,and BMMSC were identified by inducing differentiation into osteogenic,adipose,and chondrogenic tissues.Furthermore,flow cytometry was applied for identifying BMMSC by analyzing the positive rate of mesenchymal stem cell marker molecules.2.hp-BMMSC model was established by culturing BMMSC in 1%O2,5%CO2,94%N2 for 24 hours,and the proliferation of n-BMMSC,hp-BMMSC,rapa-hp-BMMSC was tested by using cell counting kit-8(CCK-8).3.n-BMMSC,hp-BMMSC,rapa-hp-BMMSC were labeled by fluorescent dye CM-Dil,and the labeled cells were infused into HIRI model rats via the portal vein respectively 12 hours after reperfusion.The liver samples were gained and observed to compare the homing ability among the different treated BMMSC to the injured liver.4.Rat models of hepatic ischemia reperfusion injury(HIRI)were constructed by blocking 70%liver blood supplies for 45 minutes.At the beginning of reperfusion,n-BMMSC,hp-BMMSC,and rapa-hp-BMMSC suspension or equivalent fresh medium was infused respectively into rats via portal vein.Serum transaminase,liver superoxide dismutase(SOD),malondialdehyde(MDA)levels,and pathological scores were measured.Interleukin-6(IL-6),interleukin-1p(IL-1p),tumor necrosis factor-a(TNF-α)expression were detected to compare the protective effects of different cells on HIRI.5.Mammalian target of rapamycin(mTOR),hypoxia inducible factor 1a(HIF-a)mRNA and protein were detected by RT-qPCR and Western Blotting to explore mechanisms of enhanced the protective of hypoxia-preconditioned BMMSC on HIRI.Results1.A large number of BMMSC were obtained from bone marrow after expanding in-vitro.BMMSC could be induced to differentiate into osteogenesis,adipogenesis,and chondrogenesis.Positive rate of MSC-specific marker molecules was confirmed by flow cytometry on BMMSC surface.All these results indicated that BMMSC in this experiment was a subset of mesenchymal stem cells.2.1%O2,5%CO2,94o N2 for 24 hours was the optimal hp-BMMSC protocol,and its proliferation was the most vigorous compared with n-BMMSC.Besides,this effect ccould be inhibited by rapa intervention.3.Under the same dose,the positive rate of hp-BMMSC homing to the injured liver was significantly higher than that of n-BMMSC.Compared with hp-BMMSC,the homing rate of rapa-hp-BMMSC was significantly decreased,which indicates that BMMSC’s enhanced homing ability may be related to the expression of mTOR.4.n-BMMSC reduced liver transaminase,liver tissue MDA and increased SOD activity,and inhibited the expression of IL-6,IL-1β and TNF-α.This effect was most obvious during the first 12 hours of reperfusion.Compared with n-BMMSC,hp-BMMSC enhanced the above hepatoprotective effects,but these effects in rapa-hp-BMMSC was significantly weakened.5.Compared with n-BMMSC,mTOR and HIF-la mRNA and protein expression in hp-BMMSC were significantly up-regulated,while these expression in rapa-hp-BMMSC were significantly down-regulated,suggesting that hp-BMMSC enhanced The protective effects of HIRI may be regulated by the mTOR-HIF-1α pathway.Conclusions1.Bone marrow is the stable and reliable source of BMMSC.Induced multi-differentiation stain and flow cytometry detection are necessary for BMMSC identification.2.hypoxia-precondition is an effective method to improve the proliferation of BMMSC and enhance its homing ability.Compared with n-BMMSC,hp-BMMSC exertes enhanced liver protecting effects in HIRI.3.hp-BMMSC may regulate the hypoxia adaptation of BMMSC via mTOR-HIF-1α signal axis,thereby improving its biological function and enhancing the liver protection effects in HIRI.4.The most intense inflammation and the heaviest damage hit the liver in the early stage of HIRI,especially during the first 12 hours.Whether n-BMMSC or hp-BMMSC,its liver protecting effects on HIRI are most obvious in this period.