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Objective To investigate the effects of cholinergic pathway on acute renal tubular cell injury induced by acute oxygen and glucose deprivation. Methods Rat kidney macrophages were isolated and cultured for constructing macrophages and renal epithelial cells co-cultivating model of oxygen-glucose deprivation (OGD), and the model cells were divided into three groups: OGD alone group, acetylcholine (ACh 100μmol/L)+OGD group and ACh + galantamine (Gal 10μmol/L)+OGD group. The cells underwent OGD treatment for 1 hour, and normally cultured for 24 hours. The expressions of TNF alpha, IL-1 beta, and IL-10 in supernatant fluid were detected by ELISA, the renal tubular cell viability was determined by MTT assay, the expression of acetylcholine esterase (AChE) mRNA and protein were determined by RT-qPCR and Western blotting. The activity of AChE was determined by colorimetric method. Results The expressions of TNF alpha (pg/ml) in OGD, Ach+OGD group, Ach+Gal+OGD groups were 140.2±44.81, 119.46±4.42 and 103.31±1.62 respectively (P0.05); The values of renal tubular cell proliferation were 55.02%±6.28%, 66.65%±6.47%, and 79.75%±4.22% respectively (P0.05); those of AchE protein were 0.66±0.07, 0.74±0.04 and 0.67±0.06 respectively (P>0.05); The activity of AChE (kU/L) was 0.51±0.02, 0.35±0.05 and 0.32±0.04 respectively (P=0.001, 0.001 and 0.368). Conclusions ACh and Gal could inhibit the secretion of inflammatory mediators and cholinesterase activity and can reduce the acute hypoxic renal tubular cell injury. The modulation of the cholinergic pathway in macrophages may be the important treatment method for acute renal injury in the future.
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Objective To evaluate the role of β-arrestin-1 in inhibition of endotoxin-induced activation of nuclear factor kappa B (NF-κB) in human pulmonary microvascular endothelial cells (HPM-VECs) by penehyclidine hydrochloride (PHC).Methods HPMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105/ml,and were randomly divided into 5 groups (n =20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (group LPS),PHC + LPS + empty plasmid transfection group (group P+LPS),LPS + β-arrestin-1 gene-shRNA transfection group (group LPS+shRNA) and PHC + LPS + β-arrestin-1 gene-shRNA transfection group (group P+LPS+shRNA).HPMVECs were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1gene-shRNA.At 24 h of incubation,PHC with the final concentration of 2 μg/ml was added,the cells were incubated for 1 h,LPS with the final concentration of 0.1 μg/ml was then added,and the cells were continuously incubated for another 1 h.The supernatant was collected to measure the activity of lactic dehydrogenase (LDH).The cell suspension was collected for determination of vascular cell adhesion molecule-1 (VCAM-1) expression and NF-κB activities and NF-κB inhibitor I-κB and β-arrestin-1expression.Results Compared with group C,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in LPS and LPS+shRNA groups.Compared with group LPS,the activities of LDH in supernatant were decreased,VCAM-1 expression was down-regulated,NF-κB activity was significantly decreased,and I-κB and β-arrestin-l expression was up-regulated in group P+LPS,and no significant change was found in the parameters mentioned above in group P+LPS+shRNA.Compared with group P+LPS,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in group P+LPS+shRNA.Conclusion PHC inhibits endotoxin-induced activation of NF-κB in HPMVECs completely through up-regulating β-arrestin-1 expression.
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Objective To evaluate the effect of nicotine pretreatment on cardiac function following myocardial ischemia-reperfusion (I/R) in rats.Methods Sixty male Sprague-Dawley rats,weighing 200-250 g,were randomly divided into 3 groups (n =20 each):sham operation group (group S),group I/R and nicotine pretreatment group (group N).The rats were anesthetized with intraperitoneal 20％ urethane 1 g/kg,tracheostomized and mechanically ventilated.Myocardial ischemia was induced by occlusion of the left anterior descending branch of coronary artery for 30 min followed by 120 min of reperfusion.The left anterior descending branch of coronary artery was only exposed,but not occluded in group S.Nicotine 400 μg/kg was injected intravenously via the right jugular vein at 30 min before myocardial ischemia in group N.The equal volume of normal saline was injected instead in groups S and I/R.Before ischemia,at 30 min of ischemia and at 30 and 120 min of reperfusion,10 rats from each group were chosen for record of left ventricular systolic pressure (LVSP),left ventricular diastolic pressure (LVDP),± dp/dtmax,HR and mean arterial pressure (MAP).Blood samples were collected from the right carotid artery of the left 10 rats in each group at 60 min of reperfusion to measure plasma CK-MB activity and cTnI and TNF-α concentrations.Results Compared with group S,MAP and LVSP at T2,3 and HR,LVDP and ± dp/dtmax at T1-3 were significantly decreased,and the plasma CK-MB activity and cTnI and TNF-α concentrations were increased in group I/R,and LVDP at T1,2 and HR and ± dp/dtmax at T1-3 were significantly decreased,and the plasma CK-MB activity and cTnI and TNF-α concentrations were increased in group N (P ＜ 0.05).Compared with group I/R,MAP,HR,LVSP,LVDP and ± dp/dtmax were significantly increased at T3,and the plasma CK-MB activity and cTnI and TNF-α concentrations were decreased in group N (P ＜ 0.05).Conclusion Nicotine pretreatment can reduce myocardial I/R injury through activating cholinergic anti-inflammatory pathway,thus improving cardiac function in rats.
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Objective To investigate the effect of nicotine on coagulation abnormalities in endotoxemic rats.Methods Ninety-six male SD rats weighing 200-250 g were randomly divided into 4 groups (n=24 each): group normal saline (group NS);group LPS;group nicotine(group NIC)and group α-bungarotoxin (α7 nicotinic acetylcholine receptor antagonist, group α-BGT) . Endotoxemia was induced by LPS 10 mg/kg injected via femoral vein in LPS, NIC and α-BGT groups. In group NIC nicotine 400 μg/kg was injected intraperitoneally at 30 min before LPS injection. In group α-BGT α-BGT 1 μg/kg was injected intraperitoneally at 15 min before intraperitoneal nicotine. Prothrombin time(PT),activated partial thromboplastin time(APTT),fibrinogen(Fib),antithrombin (AT),von Willebrand factor(vWF),plasminogen activator inhibitor-1(PAI-1),D-dimer,platelet count and TNF-α were measured before (baseline) and 2, 4 and 6 h after LPS injection.Results PT and APTT were significantly prolonged and plasma Fib and AT concentrations and platelet count were significantly decreased, while plasma PAI-1, D-dimer, vWF and TNF-α concentrations were significantly increased after LPS administration in group LPS as compared with group NS. Nitotine pretreatment significantly attenuated the LPS-induced changes in group NIC.The effect of nicotine was counteracted by α-BGT. Conclusion Nicotine can attenuate coagulation abnormalities induced by LPS by acting on α7 nicotinic acetylcholine receptor.