| Endotoxin shock (ES) is a common and severe pathological process in clinical practice, ES and multle organ dysfunction syndrome (MODS) cause most frequent mortality of ICU patient and are hard to solve problem in the practice of forensic medicine. The systemic circulation vaso-relaxation together with the manifestations of marked reduction in systemic mean arterial pressure(MAP) is characteristic of the initial stage of endotoxin shock. Lipopolysaccharide (LPS),one of the main component of G-negative bacteria, may stimulate the activation of vascular smooth muscle cells and abnormal expressions of a variety of vascular factors such as superoxide dismutase (SOD),inducible nitricoxide synthase (iNOS) together with the excess formation of free oxygen radical, nitric oxide(NO).These factors may be the most cause of vascular pressure depression at the initial stage of endotoxin shock.Cholecystokinin(CCK),a neuropeptide, is widely distributed in central nervous system,digestive system,immuno-organs cardiac and vascular system,takes part in the regulation of the different physiological and patho-physiological processes. Previous studies showed that cholecystokinin octapeptide (CCK-8),the predominant active fragment of endogenous CCK,had vascular effects in both conscious and anesthetized animals and elicited dose-dependant increase in arterial blood pressure. It was reported that intravenous administration of low dose of CCK-8 caused the constrictions of renal, mesenteric and hindquarter arteries,whereas high dose of CCK-8 caused vasodilatations of the above arteries. A series of studies in our lab have demonstrated that endogenous and exogenous CCK-8 had anti-ES effect with the ability to decrease the mortality of ES rats, enhance the activity of SOD, reduce the increase in iNOS activity and relief the diastolic abnormal reaction of aorta thoracalis incubated with LPS, reverse the fall in MAP, and prevent the pulmonary arteral hypertension(PAH),and attenuate the pathological changes of main organs. In contrast,pretreatment with proglumide,a nonselective CCK-receptor antagonist,could delay the recovery of MAP and lead to the increase of mortality of ES rats. It has been well known that CCK-8 exerts a variety of physiological actions through CCK receptors, which have been pharmacologically classified into two subtypes CCK-AR and CCK-BR according to their affinity to the peptide agonist sCCK-8 and gastrin. CCK receptors belong to G protein-coupled receptor superfamily and were extensively distributed in mammalian body. A series of studies in our lab showed that proglumide,CR-1409 (a selective CCK-A receptor,CCK-AR,antagonist), and CR-2945 ( a selective CCK-B receptor,CCK-BR, antagonist) could reverse the role of CCK-8 anti-ES effects.Nuclear factor-kappa B(NF-кB) is an important transcriptional factor which is involved in the mediation of cellular damage induced with LPS.It was reported that CCK-8 could inhibit the activity of NF-кB in the lung tissue of endotoxia and pulmonary interstitial macrophages (PIMs) induced by LPS. The signal transduction mechanisms through which LPS activates VSMCs are anfractuous. However it is undoubtful that LPS-LBP-PKC and PTK-IKK-IκB-NF-κB-kinase pathways play a pivotal role. LPS binds LPS-binding protein (LBP) to form LPS-receptor complex, resulting in the recruitment of an adapter named MyD88. The death domain of MyD88 then recruits downstream associated kinase, including NF-κB-inducing kinase (NIK),protein kinase C(PKC) and protein tyrosine kinase(PTK)to the receptor complex. Activated NIK or PKC/PTK are individually capable of activating the IκB kinase(IKK) complex. Subsequently, IκB is phosphorylated and degraded, leading NF-κB to translocate to the nucleus, and initiate the related-genes transcription.Based on the fact that VSMC is main effective cells in blood vessel and the previous studies about the anti-ES of CCK-8 in our lab, the present study was to examine⑴the expression of CCK receptor and the effect of LPS on the expression of CCK receptor in rats thoracic aortic smooth muscle cells (TASMCs),⑵effect of CCK-8 on the expression of iNOS and its mechanism,⑶participation of PTK in the regulation by CCK-8 for the enhenced NF-κB activity of TASMCs induced by LPS,⑷the signal transduction mechanism of CCK-8 effect on the expression of SOD⑸effect of CCK-8 on the expression of opioid receptor and its mechanism in ES rats.1 Receptor mechanisms underlying the regulation by CCK-8 for LPS-induced iNOS expression in rat TASMCs1. 1 The expression of CCK-receptor in rat TASMCs and the effect of LPSObjective To observe the expression of CCK-receptor and the effect of LPS on the receptor in rat TASMCs .Methods Rats TASMCs were isolated,cultured with sticking pieces method and stimulated with LPS(0.1 mg/L) in different time. The expression of CCK-AR and CCK-BR gene was assayed by reverse transcription polymerase chain reaction (RT-PCR), the CCK-AR and CCK-BR proteins were detected by immuocytochemistry technique in the presence or stimulated with LPS. 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. 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) CCK-AR/CCK-BR mRNA and protein were detected in rat TASMCs, and CCK-AR/CCK-BR mRNA amplification products had a size of approximately 1.37kb and 480bp, respectively. In each cell or tissue sample, allβ-actin amplification products were of 450bp length.(2) The relative expressed quantity (ratio of arbitrary unit overβ-actin) of CCK-AR was (0.3654±0.132)% in control group and (0.7172±0.117)%, (1.7211±0.103)%, (0.8036±0.175)%, (1.1315±0.18)%in LPS 1h, 2h, 4h and 8h, respectively, and significant increases in its expression were observed at indicate time incubated with LPS (p<0.05), and with an highest expression of which at 2h(p<0.01), with a little degression at 4h, with a returning at 8h. For CCK-BR mRNA, the relative expressed quantity was (0.9115±0.106)% in control group and (1.0424±0.094)%, (1.9741±0.194)%, (1.3329±0.062)%, (1.1599±0.108) % in LPS 0.5h, 2h, 6h and 12h, respectively, and the expression level of every time point in LPS group was higher than that of control group (p<0.05), and with an highest expression of which at 2h(p<0.01). The relative expressed quantity of CCK-BR mRNA was increased in comparison with that of CCK-AR mRNA (p<0.05).(3) The brown yellow grains, that expression of CCK-AR/ CCK-BR membrane proteins which were detected by immuocytochemistry technology , were observed by light microscope at cellular membrane and cytoplasm in control group. And the masccline cells population exceed 5%. The expression of CCK-AR/CCK-BR protein were up-regulated at 2h in TASMCs after LPS (0.1mg/L)incubation compared with control group, and the CCK-BR protein was increased in comparison with that of CCK-AR protein (according to the numbers of masccline cells in each highpower field).Conclusions The expressions of CCK-AR/CCK-BR mRNA and protein were detected in cultured rats TASMCs in vitro, and the up-regulation expression were induced by LPS. These results had offered structure basis for investigating further the signal transduction mechanism of CCK-8 anti-ES.1. 2 Receptor mechanisms underlying the regulation by CCK-8 for LPS-induced iNOS expression in rat TASMCsObjective To observe the effect of CCK-8 on iNOS expression and CCK receptor mechanism in TASMCs induced by LPS.Methods TASMCs were cultured and stimulated with LPS in the absence or presence of CCK-8 and/or CCK receptor antagonist CR-1409/ CR-2945, and the iNOS gene was assayed by RT-PCR. iNOS protein level in the cytoplasma 16h after stimulation was detected by Western blot. Optical density value was used to show the relative amount of the iNOS gene and protein. 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)The untreated TASMCs spontaneously produced too little iNOS to be detected.(2) Stimulating TASMCs with 0.1mg/L LPS resulted in an obvious increase in iNOS gene and protein (P<0.01), which were as 3.81,4.16 times as that in control group respectively.(3) Treating TASMCs with CCK-8, at the concentrations of 10-810-6 mol/L, led to a dose-dependent inhibition of iNOS gene and protein production(P<0.01) treated by LPS.(4) Pre-treating TASMCs with 10-5 mol/L CR-1409/CR-2945 rivalried the inhibitory action of CCK-8. The expressions of iNOS gene and protein increased 52.96% and 33.7%(P<0.01) respectively compared with CCK-8+LPS group pre-treated with CR-1409, the expressions of iNOS gene and protein increased 42.16%(P<0.01) and 23.56% (P<0.05) respectively compared with CCK-8+LPS group pre-treated with CR-2945. These results indicated that the antagonistic action of CR-1409/CR-2945 was living its gene level and the inhibitory action of CR-1409 to CCK-8 was powerful than CR-2945 at a same dose. iNOS level was not affected by CCK-8 (10-8mol/L),CR-1409,CR-2945 alone in comparison with the control group(P>0.05).Conclusions CCK-8 inhibited LPS-increased iNOS mRNA and protein expression in TASMCs, which was mediated through specific CCK receptors at target cellular membrane. And the affect of CCK-AR to CCK-8 was powerful than CCK-BR.2 The signal transduction underlying the regulation by CCK-8 for LPS-induced SOD expression in rat TASMCs2. 1 Involvement of PTK signaling pathway in regulation by CCK-8 for LPS- induced NF-κB activity in rat TASMCsObjective To elucidate the anti-ES mechanism of signal transduction of CCK-8. NF-кB is an important transcriptional factor in ES. PTK which act as the upstream protein of NF-κB can mediate varied of different signal transduction pathway . It is well known that CCK-8 can inhibite NF-κB activity of PIMs,the lung tissues in ES rat and ECV-304. However the effect of CCK-8 on the change of NF-κB activity and the effect of PTK in rat TASMCs has remained nuclear.Methods TASMCs were stimulated with LPS in the absence or presence of CCK-8 and/or PTK inhibiter genistein(Gen), and NF-κB binding activity was analyzed by electrophoretic mobility shift assay (EMSA) 1h after stimulation. The IκBαprotein level in the cytoplasma 30min after stimulation was detected by Western blot. The nuclear translocation of P65 protein 1h after stimulation was observed by immuocytochemistry. 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) The NF-κB binding activity was significantly higher in TASMCs stimulated with 0.1mg/L LPS in comparison with unstimulated cells (P<0.01), and additional treatment with CCK-8 markedly reduced the binding activity in a dose-dependent manner. CCK-8 at the concentrations of 10-10 mol/L, 10-8 mol/L, and 10-6 mol/L inhibited LPS-induced NF-κB binding activity by 27%, 66%, 80% respectively (P<0.05, P<0.01). Pre-treating TASMCs with 10-5 mol/L Gen , NF-κB binding activity was inhibited obviously which was significantly different from LPS and LPS+CCK group (P<0.05, P<0.01). CCK-8 or Gen alone had no effect on the NF-κB binding activity (P>0.05). The binding specificity was confirmed by using homologous (NF-κB) and nonhomologous (AP-2) oligonucleotides as competitors.(2) The IκBαprotein level in TASMCs was markedly decreased 30min after incubation with 0.1mg/L LPS (P<0.01), and CCK-8 at the concentrations of 10-1010-6 mol/L obviously increased IκBαprotein level in TASMCs stimulated by LPS (P<0.05). Pre-treating TASMCs with 10-5 mol/L Gen , the protein level increased obviously, and it was significantly different from LPS (P<0.05), but was not significantly different from LPS+CCK (P>0.05) . CCK-8 or Gen alone had no effect on the IκBαprotein level (P>0.05).(3) The p65 protein expression of NF-κB was strong in cytoplasm of unstimulated TASMCs in where there are a great quantity of brown yellow grains, but the expression was very weak in cytoblast of TASMCs by immuocytochemistry. The p65 protein expression was significantly higher in cytoblast and nuclear translocation stimulated with 0.1mg/L LPS for 1h as compared with unstimulated cells (P<0.01), and pre-treatment with 10-1010-6 mol/L CCK-8, in a dose-dependent manner, markedly reversed the enhancement of p65 protein expression induced by LPS(P<0.01). Pre-treating TASMCs with 10-5 mol/L Gen , the p65 protein expression in cytoplasm was inhibited obviously which was significantly different from LPS and LPS+CCK group (P<0.05, P<0.01). CCK-8 or Gen alone had no effect on the NF-κB nuclear translocation (P>0.05).Conclusions CCK-8 inhibited NF-κB activity through PTK induced by LPS in rat TASMCs, which might be one of the upstream mechanism of CCK-8 anti-ES.2. 2 The signal transduction underlying the regulation by CCK-8 for LPS-induced SOD expression in rat TASMCsObjective Superoxide dismutase (SOD) is a most important antioxidase which can eliminate superoxide anion. Reduced MAP and vasodilatation of systemic circulation were associated with the oxyradical production during ES. There is accumulating evidence that CCK-8 possesses the effect of anti-ES, can reverse the depression of SOD activity, inhibit the increase in Cu-Zn SOD/Mn SOD mRNA expression induced by LPS. CCK-8 inhibited NF-κB activity through PTK induced by LPS in rat TASMCs. However, the signal transduction underlying the regulation by CCK-8 for LPS-induced SOD expression in rat TASMCs is unclear. NF-кB is a significant factor which is involved in production of free radical in TASMCs incubated with LPS.Methods TASMCs were stimulated with LPS in the presence or absence of CCK-8 and/or PTK inhibitor genistein(Gen), Cu-Zn SOD/Mn SOD mRNA was analyzed by RT-PCR 4h after stimulation. 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) The basal expression of Cu-Zn SOD/Mn SOD mRNA were detected in unstimulated cells;(2) Cu-Zn SOD/Mn SOD mRNA significantly up-regulated in TASMCs stimulated with 0.1mg/L LPS in comparison with unstimulated cells (P<0.05);(3) Pre-treatment with CCK-8(10-8mol/L) markedly reduced the expression of Cu-Zn SOD/Mn SOD mRNA (P<0.01) stimulated with LPS;(4) Pre-treating TASMCs with 10-5 mol/L Gen , the expression of Cu-Zn SOD/Mn SOD mRNA was inhibited obviously, and it was significantly different from LPS (P<0.01), but was not significantly different from LPS+CCK (P>0.05) ;(5) CCK-8 or Gen alone, the expression of Cu-Zn SOD/Mn SOD mRNA down-regulated obviously which was significantly different from control group (P<0.05).Conclusions CCK-8 inhibited the increasion of Cu-Zn SOD/Mn SOD mRNA expression through PTK induced by LPS in rat TASMCs, which might be one of the signal transduction mechanism of CCK-8 anti-ES.3 Molecular mechanisms underlying the regulation by CCK-8 for LPS-induced opioid receptor mRNA expression in rat TASMCs3. 1 Effect of CCK-8 and its receptor on opioid receptor mRNA expression in rat TASMCs stimulated by LPSObjective To elucidate the molecular mechanism of CCK-8 anti-ES.Opioid receptors(OR) include multi-subtypes such as .. , .. .. .. .. . and are extensively?distributed and can mediate wide processes of physiology and pathology with opioid peptide. It was reported that the vascular wall of human and animal exist opioid receptors and OR might participate the course of hemorrhagic shock; naloxone, a non-specificity opioid receptor antagonist, can express anti-ES. Does the OR exist in rat TASMCs? And the effect of CCK-8 and its receptor on -OR mRNA expression stimulated by LPS is unclear.Methods TASMCs were cultured and stimulated with LPS in the presence or absence of CCK-8 and/or CCK receptor antagonist CR-1409/CR-2945,Nor-BNI, the -OR mRNA was assayed by RT-PCR 16h after stimulation. 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) The basal expression of -OR mRNA were detected in unstimulated cells;(2) The -OR mRNA significantly up-regulated in TASMCs stimulated with 0.1mg/L LPS in comparison with unstimulated cells (P<0.01);(3) Pre-treatment TASMCs with CCK-8, at the concentrations of 10-810-6 mol/L, led to a dose-dependent inhibition of -OR mRNA treated by LPS(P<0.05);(4) Pre-treating TASMCs with Nor-BNI, the expression of -OR mRNA was inhibited obviously, and it was significantly different from LPS/LPS+CCK group(P<0.05); but was not significantly different from LPS+CCK (P>0.05) ;(5) Pre-treating TASMCs with 10-5 mol/L CR-1409/CR-2945 rivalried the inhibitory action of CCK-8, and the rivalring of CR-2945 was significantly different from LPS+CCK group (P<0.05), the rivalring of CR-1409 was not significantly different from LPS+CCK group (P>0.05);(6) CCK-8,CR-1409,CR-2945 or Nor-BNI alone, the expression of -OR mRNA up-regulated slightly which was not significantly different from control group (P>0.05). Conclusions CCK-8 inhibited the increasion of -OR mRNA expression through its receptor induced by LPS in rat TASMCs, which might be one of the mechanism of CCK-8 anti-ES.3. 2 Molecular mechanisms underlying the regulation by CCK-8 for LPS-induced opioid receptor mRNA expression in rat TASMCsObjective To elucidate the mechanism of signal transduction of CCK-8 anti-ES. Protein kinase C(PKC) is a family of serine/ threonine protien kinase which is composed by more than ten members. PKC that exist extensively in tissues,organs and cells can regulate the growth,metabolism,proliferation and differentiation of cells. It was confirmed that CCK-8 inhibited the increase in -OR mRNA expression in rat TASMCs induced by LPS, However, the signal transduction underlying the regulation by CCK-8 for LPS-induced -OR mRNA expression in rat TASMCs is unclear.Methods TASMCs were stimulated with LPS in the presence or absence of CCK-8 and/or PKC inhibitor chelerythrine (CHE), morphine(Mor), -OR mRNA expression was analyzed by RT-PCR 16h after stimulation. 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) Pre-treating TASMCs with CHE, the expression of -OR mRNA was inhibited obviously, and it was significantly different from LPS/LPS+CCK group(P<0.01,P<0.05);(2) Pre-treating TASMCs with Mor, the expression of -OR mRNA was inhibited partly on treated by CCK-8, which was significantly different from Mor group (P<0.05), the inhibition of CCK-8 to -OR mRNA was relieved on treated by LPS, which was significantly different from LPS+CCK/CCK+Mor group (P<0.05);(3) CHE alone, the expression of -OR mRNA up-regulated slightly which was not significantly different from control group (P>0.05), and Mor alone, the expression of -OR mRNA up-regulated significantly which was significantly different from control group (P<0.01).Conclusions CCK-8 inhibited the increasion of -OR mRNA expression through PKC induced by LPS in rat TASMCs, which might be one of the signal transduction mechanism of CCK-8 anti-ES.CONCLUDIONSIn the present study the modular effect of CCK-8 on LPS-induced the activation of TASMCs and the related signal transduction mechanism were systematically investigated from receptors to transcription factors.New findings were as follows.1. There was the presence of both CCK-AR and CCK-BR mRNA and protein expression in rat TASMCs. CCK-AR and CCK-BR mRNA and protein expression could be obviously up-regulated by LPS, in which the relative expressive quantity of CCK-BR mRNA was higher than that of CCK-AR mRNA.2. CCK-8 dose-dependently inhibited LPS-induced iNOS expression through CCK receptors in rat TASMCs.3. CCK-8 dose-dependently inhibited NF-κB activity through PTK in the rat TASMCs induced by LPS.4. CCK-8 inhibited Cu-ZnSOD and MnSOD mRNA expression in the rat TASMCs induced by LPS in which LPS-PTK-NF-κB-SOD signaling pathway was involved.5. CCK-8, through CCK receptors, inhibited the up- expression?of? -OR mRNA in rat TASMCs induced by LPS, in which LPS-PKC-OP signaling pathway was involved.
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