| Aim: To explore the effects of heme oxygenase-1(HO-1)-modified bone marrow mesenchymal stem cells(BM MSCs)on the microcirculation and energy metabolism of hepatic sinusoids following reduced-size liver transplantation(RLT)in a rat model.Methods: BM MSCs were isolated and cultured in vitro using an adherent method,identified by flow cytometric analysis and transformation,then transduced with HO-1-bearing adenovirus to construct HO-1/BM MSCs.An acute rejection model following 50 % RLT was established in rats using a two-cuff technique,with recipients divided into three groups receiving treatment with normal saline(NS),BM MSCs or HO-1/BM MSCs,NS,BM MSCs and HO-1/BM MSCs were injected through the superficial dorsal veins immediately after RSL,recipients were observed for six time points.The histopathological changes in the liver tissues were observed by HE staining,liver function was detected biochemically,the levels of endothelin-1(ET-1),endothelial nitric-oxide synthase(e NOS),inducible nitric-oxide synthase(i NOS),nitric oxide(NO),and hyaluronic acid(HA)were detected using ELISA.The portal venous pressure(PVP)was detected by Power Lab.The expressions of ET-1,i NOS,e NOS,and Von Willebrand factor(v WF)protein in the transplanted liver were detected using immunohistochemistry and western blotting.ATPase in the transplanted liver was detected using chemical colorimetry,and the ultrastructural changes were observed using a transmission electron microscope.Results: By isolating bone marrow from rats femur and tibia,cell adherence,we can successfully obtain BM MSCs which were characterized by high quantity and purity.An acute rejection model following 50% RLT in rats established by two-cuff technique is successful and stable.The serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the three groups increased initially and then decreased with increasing post-operative time,while total bilirubin(TBIL)tended to increase with time;the serum liver enzymes and TBIL of the HO-1/BM MSCs group were significantly lower than those of the BM MSCs and NS groups on POD1,5,7,and 14(P < 0.05).As for the microcirculation of hepatic sinusoids:(1)Rejection was significantly severe in NS groups on POD7,in this time point BM MSCs group and HO-1/BM MSCs group were significantly ininhibiting rejection and reparing lesion,futherly measured PVP,PVP decreased significantly in HO-1/BM MSCs(10.67 ± 0.35 mm Hg)and BM MSCs groups(10.95 ± 0.22 mm Hg)compared with that of the NS group(21.26 ± 0.20 mm Hg)(P < 0.01);and the decreasing PVP effects of HO-1/BM MSCs were superior to that of simple BM MSCs.(2)HO-1/BM MSCs could inhibit the expressions of ET-1,promote the expressions of e NOS and inhibit the expressions of i NOS,inhibit amounts of NO,and maintain the equilibrium of ET-1/NO(P < 0.05);the effects of HO-1/BM MSCs were superior to that of simple BM MSCs.The level of ET-1 in the transplanted liver decreased initially and then increased as the post-operative time extended,the level of ET-1 in the HO-1/BM MSCs group was the significantly lower on POD1,5,7,and 14(P < 0.05)compared with other two groups.The level of e NOS in the transplanted liver tended to increase with increasing post-operative time,the level of e NOS was significantly higher in HO-1/BM MSCs group on POD 1,5,7,and 14(P < 0.05)compared with other two groups.The level of i NOS in the transplanted liver tended to increase with the extension of post-operative time,the level of i NOS in the HO-1/BM MSCs was significantly lower than other two groups on POD1,7,and 14(P < 0.05).The serum expression of NO tended to increase initially and then decrease with increasing post-operative time;however,the decreasing tendency was not significant in all groups,the serum NO in the HO-1/BM MSCs group was significantly higher than other two groups on POD1(P < 0.01).The serum NO on POD 5 and 7 was significantly lower in the HO-1/BM MSCs group compared with NS group(P < 0.01).(3)HO-1/BM MSCs promoted the expression of v WF in hepatic sinusoidal endothelial cells(SECs),and promoted the degradation of HA,compared with those of the NS group and BM MSCs group(P < 0.05).Intrahepatic expression of v WF tended to increase initially and then decrease after POD 7;however,NS group showed no significant increasing trend,the expression of v WF in the HO-1/BM MSCs group was significantly higher than other two groups on POD 1,5,7and POD 14(P < 0.05).The serum HA in all groups tended to increase initially,then decrease,and later increase again with increasing post-operative time,serum HA was significantly lower in the HO-1/BM MSCs group compared with other two groups at all time points(P < 0.01).For the energy metabolism of the transplanted liver: the damage was more severe on POD7 in the NS group,with obvious mitochondrial swelling,vacuolization,and disturbed structures of mitochondrial cristae,some even disappeared.Mitochondria in the HO-1/BM MSCs and BM MSCs groups showed mild swelling,no vacuolization,and more integrated structures of the mitochondrial ridge.Mitochondrial AST(ASTm)howed a tendency to increase initially and then decrease with increased post-operative time.The expression of ASTm was lowest in the HO-1/BM MSCs group and highest in the NS group on POD0,1,5,7 and 14(P < 0.05).The activities of ATPase in the transplanted liver tended to increase initially and then decrease after POD 7 as the post-operative time increased,the activities of ATPase in the HO-1/BM MSCs group were higher than those of the BM MSCs and NS groups on POD1,7,and 14(P < 0.05);the activities of ATPase in the HO-1/BM MSCs and BM MSCs groups were both higher than that in NS group on POD5(P < 0.05).HO-1/BM MSCs could repair the damaged mitochondria,reduce the level of mitochondrial aspartate aminotransferase(m AST)and improve activity of ATPase,compared with the other two groups(P < 0.05).The effects of HO-1/BM MSCs were superior to that of simple BM MSCs.Conclusions: The protective effects of HO-1/BM MSCs for liver with acute rejection in rats following RLT could act as follows: 1.It could reduce the expression of ET-1,promote the generation of NO the improve ET-1/NO balance,decrease PVP,and reduce apoptosis of SECs,all of those effcets significantly improved the microcirculation of hepatic sinusoids.2.HO-1/BM MSCs improved liver energy metabolism by imparing the function of mitochondria in damaged hepatocytes,and enhancing activities of ATPase in rats following RLT.3.The effects of HO-1/BM MSCs were superior to that of simple BM MSCs on improving microcirculation of hepatic sinusoid and energy metabolism in rats following RLT. |
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