Investigation On The Roles Of AngⅡ, MAPK And NF-κB In The Splenic Vein Angiopathy Of Portal Hypertension Patients

Objective: My project aims to investigate the expression levels of local angiotensin II (AngII) and its receptors, mitogen-activated protein kinase(MAPK) and nuclear factor-kappa B(NF-κB) in splenic vein of portal hypertension (PHT) patients. The roles AngII, MAPK and NF-κB in the proliferation of human Splenic vein Vascular Smooth Muscle Cel(lSvVSMC)of PHT patients will also be investigated. In the end, the effects of MAPK and NF-κB on the proliferation of human SvVSMC of portal hypertension induced by angiotensin II.Method: Twenty-six patients with posthepatitic cirrhosis portal hypertension(18 males and 8 females) admitted to the First Affiliated Hospital of Fujian Medical University were subjected to splenectomy plus pericardial devascularization during hospitalization served as PHT group, and 10 patients(7 males and 3 females) with traumatic spleen rupture subject to splenectomy during the same period as the control group. Radioimmunoasasy (RIA) was used to determine the AngII level in Splenic Vein. The mRNA expression of AT1-RmRNA,AT2-R in splenic vein was detected by RT-PCR. The protein expression of NF-κB(p65), ERK(p-erk 42/44) and P38(phospho-p38) in Splenic Veins was determined by immunohistochemistry and western blot. SvVSMC were obtained from human splenic vein of PHT patients. WST-1 was used to measure the proliferation activity of VSMC. The protein expression of NF-κB, ERK and P38 in SvVSMC was assayed by western blot.Result:Part I: Compared to the control group, in the splenic veins of the PHT group, the expression level of local AngII was significantly higher (248.91±48.31) ng/L vs (143.35±36.45) ng/L(P<0.01), the protein expression of NF-κB, P38 and ERK was stronger (P<0.01) and the mRNA level of AT2-R increased (P<0.01), whereas the mRNA level of AT1-R dramatically reduced (P<0.01).Part II1. AngII(10^-8-10^-6mol/L) increased NF-κB activity in SvVSMC in a dose-dependent manner.2. AngII increased the protein expression of NF-κB in human PHT SvVSMC, which peaked 30 minutes after stimulation. Within the range of 10^-8-10^-6mol/L, AngII increased the proliferation activity (WST-1) of SvVSMC with markedly statistics difference (P<0.05) compared with control.3. AngII increased the protein expression of ERK in human PHT SvVSMC, which peaked 10 minutes after stimulation. Within the range of 10^-8-10^-6mol/L, AngII increased ERK activity in human PHT SvVSMC in a dose-dependent manner. Compared with control, AngII(10^-8-10^-6mol/L) increased the proliferation activity of SvVSMC with markedly statistics difference(P<0.05)4. AngII increased the protein expression of p38 in human PHT SvVSMC, which peaked 5 minutes after stimulation. Within the range of 10^-8-10^-6mol/L,, AngII(10^-8-10^-6mol/L) increased P38 activity in SvVSMC in a dose-dependent manner. AngII(10^-8-10^-6mol/L) increased the proliferation activity (WST-1) of human PHT SvVSMC with markedly statistics difference(P<0.05 or P<0.01) compared with control5. Compared to the AngII (10^-7mol/L) only treatment, the addition of the selective NF-κB antagonist PDTC(100umol/L), the selective ERK antagonist PD98059(100umol/L), the selective p38 antagonist SB202190 (10^-7mol/L) and the selective JNK antagonist SP600125(100umol/L)decreased the proliferation of SvVSMC stimulated by AngII (P<0.01). Blocked with PD98059 or PDTC,the proliferation of SvVSMC stimulated by AngII compared with control group demonstrated no significant difference (P>0.05). However, blocked with SB202190 or SP600125, the proliferation of SvVSMC stimulated by AngII(10^-7mol/L), compared with control group still illustrated significant difference ( P<0.05).6. Compared to the AngII (10^-7mol/L) only treatment, in SvVSMC stimulated by AngII (10^-7mol/L), the addition of PD98059 ( 100umol/L ) , SB202190(10^-7mol/L) and SP600125(100umol/L)decreased the activity of NF-κB (P<0.01), whereas PDTC decreased the activity of P38 (P<0.01).Conclusion:Part I: During PHT,LRAS in splenic vein was activated and the expression of NF-κB, ERK and P38 increased. Therefore, LRAS, MAPK, NF-κB may be critical for the development of the pathological changes of PHT.Part II: AngII can stimulate the proliferation of human PHT SvVSMC. AngII can activate NF-κB,ERK,P38 in the SvVSMC. In the proliferation of SvVSMC induced by AngII, NF-κB and ERK pathways are more important than P38 and ERK pathway. Activation of MAPK in the SvVSMC by AngII activates NF-κB. Activation of NF-κB in the VSMC by AngII activates p38, but not ERK. AngII may be critical for the formation and maintenance of PHT because during PHT, AngII may participate in the development of the pathological changes of the veins by activating the MAPK and NF-kB signaling pathways, which will lead to a series of subsequent effects such as the proliferation of the vascular smooth muscle cells.