The Mechanisms Of The Gene Expression Of Alpha-adrenoceptor Down Regulation In Septic Shock Rats And The Therapeutic Effects Of Antioxidants

Part 1 Protective effects of antioxidant against in the downregulation ofα₁-adrenoceptor during endotoxic shockObjective To investigate whether ratα₁-adrenergic receptor subtypes (α₁-ARs) mRNA expression are changed during septic shock and the therapeutic effect of antioxidants.Methods Forty male Sprague-Dawley rats were randomly divided into five groups: (1) the control group (n = 8); (2) lipopolysaccharide (LPS) group: LPS 15 mg·kg^-1 iv (n = 8); (3) propofol treated group: 1h after LPS, propofol 10 mg·kg^-1 iv, then continuous infusion of propofol 10 mg·kg^-1·h^-1 (n = 8); (4) uric acid treated group: 1 h after LPS uric acid 200 mg·kg^-1 ip (n = 8), (5) Melatonin treated groups: 1 h after LPS melatonin 10 mg·kg^-1 ip (n = 8), All rats were killed 6 h after LPS injection, total RNA from aorta, vein, heart, liver, lung and kidney were extracted, andα_1A-AR,α_1B-AR,α_1D-AR mRNA andβ-actin mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). Results The mRNA ofα_1A-AR,α_1B-AR in all of the organs andα_1D-AR mRNA in aorta, liver, lung and kidney was strongly down-regulated after injection of LPS(p < 0.01). In propofol treated group,α_1A-AR mRNA in the lung and kidney andα_1B-AR mRNA in all of the organs andα_1D-AR mRNA in aorta, liver, lung and kidney expressed increasing significantly comparing with the LPS group(p < 0.05). In uric acid treated group,α_1A-AR mRNA in kidney,α_1B-AR mRNA in all of the organs except lung andα_1D-AR mRNA in aorta, liver, lung and kidney expressed higher than in LPS group(p < 0.05).Conclusion Circulation disfunction in endotoxic shock is related to the down-regulation ofα₁-adrenergic receptors gene expression. Circulation disfunction during endotoxic shock being modifying by antioxidants may partly due to which reverse the down-regulation ofα₁-adrenergic receptors geneexpression.Part 2 Nitric oxide down-regulate [alpha]1-adrenoceptor gene express in rat vascular smooth muscle cellsObjective To investigate the changes ofα₁-adrenergic receptor subtypes in gene level during inflammatory response and its possible mechanisms.Methods Smooth muscle cells (SMCs) in serum-free medium were incubated for 12 hours with one of the following: (1) medium (control); (2) tyrosine (Tyr, 250μmol/L); (3) 3-nitrotyrosine(3-NT, 250μmol/L); (4) S-nitroso-N-acetylpenicillamine (SNP, 500μmol /L);(5) a mixture of TNF-αand IL-1β(Cytokines,TNF-α100 ng/ml, IL-1β50ng/ml); (6) Nω-nitro–L-arginine methyl ester with a mixture of these cytokineskines(L-NAME, 5 mmol/L); (7) melatonin with a mixture of these cytokineskines (MLT, 1 mmol/L). Total RNA from SMCs was extracted andα_1A-AR,α_1B-AR andα_1D-AR mRNA as well asβ-actin mRNA were measured by reverse transcription polymerase chain reaction(RT-PCR). The mRNA values are related to signals obtained forβ-actin mRNA.Results Comparied with the control group, Incubation of cultured SMCs with 3-NT resulted in diminishedα_1B-AR andα_1D-AR gene express(p < 0.01), and mRNA level ofα_1A-AR,α_1B-AR andα_1D-AR all decreased after incubation with SNP (p < 0.05), but there was no significant diference in the mRNA expression of the threeα₁ adrenoceptor subtyoes between the Tyr group and the normal conrol group(p > 0.05). Incubation of the cells with the mixture of cytokines also caused downregulations ofα_1A-AR andα_1D-AR mRNA(p < 0.01), whereas L-NAME or melatonin attenuated these downregulations(p < 0.05).Conclusion Nitric oxide(NO) and NO metabolism derivant 3-NT significantly attenuatesα₁-ARs gene expression in SMCs during inflammatory response, this is one of possible pathophysiological mechanisms of vascular hyporeactivity during endotoxic shock. The use of antioxidants and depression of NO synthesis may partly reduce this pathological response.Part 3 Antioxidant inhibit gene expression of induce nitric oxide synthase in endotoxic shock ratsObjective To investigate the effect of antioxidant on the mRNA expression of induce nitric oxide synthase(iNOS)in rats during endotoxic shock.Methods Forty male Sprague-Dawley rats were randomly divided into five groups: (1) the control group (n = 8); (2) lipopolysaccharide (LPS) group: LPS 15 mg·kg^-1iv (n = 8); (3) propofol treated group: 1h after LPS, propofol 10 mg·kg^-1 iv, then continuous infusion of propofol 10 mg·kg^-1·h^-1 (n = 8); (4) uric acid treated group: 1 h after LPS uric acid 200 mg·kg^-1 ip (n = 8), (5) Melatonin treated groups: 1 h after LPS melatonin 10 mg·kg^-1 ip (n = 8), All rats were killed 6 h after LPS injection, total RNA from aorta, vein, heart, liver, lung and kidney were extracted, and iNOS mRNA andβ-actin mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). The concentration of plasma malondialdehyde (MDA) and nitrate/nitrite were assayed in all groups 6 h after LPS injection.Results The mRNA of iNOS in all of the organs was strongly down-regulated after injection of LPS(p < 0.01). In propofol treated group, iNOS mRNA expression decreased 26% in aorta(P < 0.05)and was reduced more strikingly in the other organs especially in the vein comparing with the LPS group(P < 0.01). In uric acid treated group, the expressions of iNOS mRNAs were downregulated in all of the organs(P < 0.01), and in the vein iNOS mRNA expression was decreased 56.6% comparing with LPS group. Compared with LPS group, the expressions of iNOS mRNAs in all of the organs were declined significantly(P < 0.05). The plasma concentrations of MDA and nitrate/nitrite were much lower in all treated groups contrast to the endotoxic shock group(P < 0.05).Conclusion Circulation disfunction in endotoxic shock is related to the up-regulation of iNOS mRNA expression and the over production of NO. The antioxidants of propofol, uric acid and melatonin efficiently modify the vascular dysfunction by depressing the over expression of iNOS mRNA and decreasing the superoxide production in endotoxic shock rats.