Study Of The Promotive Mechanisms Of MTOR Signaling Pathway On The Development Of Portal Hypertensive Splenomegaly And The Effects Of Targeted Blockage

Part ? Relationship between splenomegaly development kinetics and m TOR signaling expression pattern in spleen of portal hypertensionAims: To explore the splenomegaly development kinetics and the m TOR signaling expression pattern in splenomegaly induced by various etiologies,as well as analyzing their relationship.Methods: Portal hypertensive(PHT)spleen samples were obtained from liver cirrhotic patients that underwent splenectomy(PHT group),and healthy spleen samples from non-portal hypertensive patients(NON-PHT group).PHT was induced by common bile duct ligation(BDL)or partial portal vein ligation(PPVL)in rats,while rats underwent sham operation(SHAM)served as control group.Rats were sacrificed on days 1,3,7,14,21.Pathological disorders were analyzed by histomorphometry.Spleen size was evaluated by spleen index that calculated by splenic weight to body weight.Western blot assay was used to examine the expression level of m TOR signaling pathway by detecting p70S6 K,S6,4E-BP1 protein expression,which are its down-stream markers.Results: The average spleen size in PHT group was significantly greater than that of NON-PHT group.The white pulp area is similar in size between the two groups,but Ki67-positive cells and ?-SMA-positive area in PHT group were significantly greater than that in NON-PHT group(p<0.01),which mostly localized in while pulp area and red pulp area,respectively.Significant increased protein expression of P-p70S6 K and P-S6 relative to their total proteins in spleens of PHT group compared with NON-PHT group were detected by western blot(p<0.05).Spleen size increased time-dependently in both rat models of PHT.Relative P-p70S6 K and P-S6 protein expression elevated gradually from time to time,which represented the progressive activation of m TOR signaling pathway.Conclusions: Splenomegaly could be the result of overall increase in the absolute splenic lymphoid tissue as a consequence of cellular proliferation and increased neovascularization in the splenic red pulp.Splenomegaly developed basically in parallel with the tendency of the activation of m TOR signaling pathway(m TOR/p70S6K/S6 stream),by which contributed to the enlargement of spleen.Part ? The effects of rapamycin by blocking m TOR signaling pathway on portal hypertensive splenomegaly and its regulatory mechanismsAims: To investigate the effects of rapamycin by blocking m TOR signaling pathway on portal hypertensive splenomegaly and its regulatory mechanisms.Methods: Portal hypertension(PHT)was induced by common bile duct ligation(BDL)or partial portal vein ligation(PPVL)in rats,while rats underwent sham operation(SHAM)served as control group.Rapamycin(2 mg·kg-1day-1)or vehicle(5% dimethyl sulfoxide solution)was administered to rats by intraperitoneal injections for a 2-week period,starting one week after operation.Measurement of portal pressure was recorded by a multichannel computer based recorder.Spleen size was evaluated by spleen index that calculated by splenic weight to body weight.Automatic hematology analyzer was used to analyze blood count.Pathological disorders were analyzed by histomorphometry.Western blot assay was used to examine the expression level of m TOR signaling pathway,angiogenic,inflammatory and apoptotic markers.Results: Morphometric quantitative analysis showed a significant larger white pulp area in BDL-VEH and PPVL-VEH rats than that in SHAM-VEH animals(p<0.05),with abundant Ki67-positive cells in the enlarged splenic lymphoid tissue.The VEGF-positive,?-SMA-positive area and Ki67-positive cells were pronouncedly upregulated in the splenic red pulp of BDL-VEH and PPVL-VEH rats,where it was consistent with sinusoidal endothelial cells(p<0.05).By immunoblotting,robust increase in the expression of VEGF and ?-SMA in spleens of BDL-VEH and PPVL-VEH rats were also detected.As evidenced by Masson staining,significant increases in fibrillar collagen were presented in splenic parenchyma of BDL-VEH and PPVL-VEH rats compared with SHAM-VEH animals(p<0.05).Proinflammatory cytokines,IL-1?,NF-?B and TNF-?,were upregulated in spleens of PHT rats.After 2-week treatment of rapamycin,the elevated P-p70S6 K and P-S6 protein expression relative to their total proteins in PHT rat spleens,as well as relative P-4E-BP1 protein expression,were strongly suppressed.Consequently,the increased portal pressure and spleen size in PHT rats were profoundly attenuated by rapamycin.Splenomegaly was markedly ameliorated by limiting lymphocytes proliferation,angiogenesis,fibrogenesis and inflammation in rats with PHT.Conclusions: The activation of m TOR signaling pathway contributed to the enlargement of spleen by promoting processes such as proliferation of lymphocytes,angiogenesis,fibrosis and inflammation.m TOR blockade by rapamycin can effectively ameliorate splenomegaly.