Regulatory Effect Of CCK-8 On LPS-induced Cu Zn-SOD MRNA Expression In Rat Vascular Smooth Muscle Cells

Objective: Endotoxin shock is a common and severe pathological process with high mortality,and has been one of the most important topics in biomedicine. The disorders of vascular regulations are characteristic of endotoxin shock with the manifestations of marked reduction in systemic mean arterial pressure whereas pulmonary arterial hyper- tension.Excessive oxygen-derived free radicals(OFR) in- duced in the process of endotoxin shock are associated with the disorders of vascular regulations.In aerobic cells,OFR are constantly produced and buffered by antioxidant defenses including the enzymes superoxide dismutase(SOD),catalase (CAT),and glutathione peroxidase(GSH-Px).However,im- balance between prooxidant and antioxidant defenses results in oxidative stress associated with the oxidative modification of biomolecules such as lipids,proteins, and nucleic acids. Superoxide anion( ),one of the OFR, could not only turn to the other OFR,but also react with nitric oxide(NO) and lead to the formation of peroxynitrite(ONOO-),while inducible nitric oxide synthase (iNOS) is also induced and lead to an increase in production of NO.ONOO- contributes to more serious cell damage and the reduction of contraction response of aortic arteries in vitro in a dose-dependent manner, demonstrating that ONOO- plays an important role in the disorders of vascular regulations in the pathogenesis of endotoxin shock.Cholecystokinin octapeptide(CCK-8), a kind of neuro- peptides,exerts a variety of physiological effects through CCK receptors, the latter of which have been classified into two subtypes CCK-AR and CCK-BR according to their affinity for CCK-8.CCK receptors belong to G protein- coupled receptor (GPCR) superfamily and are extensively distribute in mammalian animal.It was demonstrated that CCK-8 have the effect of remedy for endotoxin shock,but the underlying mechanism is not clear. Previous studies showed that CCK-8 could inhibit the production of ONOO- in pulmonary artery endothelial cells induced by LPS in vitro and had no effect on exogenous ONOO-,impling that the target of CCK-8 maybe NOS, SOD,and producted in.Our laboratory has demonstrated the regulatory effect of CCK-8 on LPS-induced changes of SOD activity in rat vascular smooth muscle cells(TASMCs). The purpose of our study is to investigate the regulatory effect of CCK-8 on LPS-induced Cu Zn-SOD mRNA expression in TASMCs.Methods: The TASMCs were isolated from male healthy Wistar rats (120±20g) in aseptic conditions,and cultured and passaged to logarithm growth phase(5 to 8 generation).1×107 cells per bottle were treated with serum- free DMEM or other reagents. The groups were decided as follows: 1. Effect of LPS on Cu Zn-SOD mRNA expression in TASMCs: The cells were stimlated respectively with 0.1mg/L LPS for 2h,4h or 8h or with NS alone,and stimlated with LPS(0.01mg/L, 0.1mg/L or 1.0mg/L) or NS for 4h. TASMCs Cu Zn-SOD mRNA expressions were determined. 2. Effect of CCK-8 on LPS-induced the changes of Cu Zn- SOD mRNA expression in TASMCs: The cells were stimulated with LPS(0.1mg/L) in the presence or absence of CCK-8 (10-10mol/L～10-6mol/L) or with NS alone. TASMCs Cu Zn-SOD mRNA expressions were determined.3. Effect of CR-1409(the specific antagonist of CCK-AR) and CR-2945 (the specific antagonist of CCK-BR)on the CCK-8 inhibited Cu Zn-SOD mRNA expression in LPS-induced TASMCs: TASMCs were stimulated with NS,LPS(0.1mg/L), CCK-8 (10-8mol/L),CR-1409(10-5mol/L) and CR-2945 (10-5mol/L) respectively or in combination.TASMCs Cu Zn-SOD mRNA expressions were determined.Reverse transcription polymerase chain reaction (RT-PCR) assay was used to examine Cu Zn-SOD mRNA expression in TASMCs. Jiangsu Jetta electrophoresis gel analysis software was used for semi-quantitative analysis of band, the absorbance ratio of Cu Zn-SOD andβ-actin was represented mRNA expression relative levels. Data were presented as x±s and analyzed with ANOVA and least significant difference (LSD) using SPSS statistical program. A level of P<0.05 was considered statistically significant.Results: 1. Effect of LPS on Cu Zn-SOD mRNA expression in TASMCs: (1) Effect of LPS on Cu Zn-SOD mRNA expression in a time-dependent manner:The untreated TASMCs spontaneously expressed a little Cu Zn-SOD mRNA.Weak increase in Cu Zn-SOD mRNA expression was detected in the TASMCs stimulated with 0.1mg/L LPS for 2h, and an obvious increase could be detected for 4h(P<0.01), and the Cu Zn-SOD mRNA continued high expression for 8h (P<0.05).(2)Effect of LPS on Cu Zn-SOD mRNA expression in a dose-dependent manner: The untreated TASMCs spontaneously expressed a little Cu Zn-SOD mRNA. Incu- bation of TASMCs with LPS(0.01mg/L, 0.1mg/L or 1.0mg/L) for 4h,changes of increase in Cu Zn-SOD mRNA expression were showed.An obvious increase could be detected in the TASMCs stimulated with 0.1mg/L LPS (P<0.01), and the Cu Zn-SOD mRNA continued high expression with 1.0mg/L LPS (P<0.05).2. Effect of CCK-8 on LPS-induced the changes of Cu Zn-SOD mRNA expression in TASMCs: Treated TASMCs with CCK-8 led to a dose-dependent inhibition of Cu Zn-SOD mRNA express- ion(P<0.01),and CCK-8 alone led to reduction in basic expression of Cu Zn-SOD mRNA(P>0.05). 3. Effect of CR-1409 and CR- 2945 on the CCK-8 inhibited Cu Zn-SOD mRNA expression in LPS-induced TASMCs: After pretreated TASMCs with CR-1409 and CR-2945, expressions of Cu Zn- SOD mRNA were markedly increased as compared with that of CCK-8+LPS group (P<0.01),but still decreased as compared with that of LPS group. The antagonism effect of CR-2945 was slightly stronger than that of CR-1409.Conclusion:The basic expression of Cu Zn-SOD mRNA in untreaed TASMCs was founded.LPS induced the increase of Cu Zn-SOD mRNA expression in TASMCs. CCK-8 inhibited the Cu Zn-SOD mRNA expression in TASMCs stimulated by LPS in a dose-dependent manner, whereas CCK-8 alone led to reduction in basic expression of Cu Zn-SOD mRNA.Both of CCK-AR and CCK-BR were involved in the mediation of the inhibitive regulation by CCK-8 for LPS-induced Cu Zn-SOD mRNA expression in TASMCs,in which mediated effects of CCK-BR was a little stronger than that of CCK-AR.