Research On The Expression Of Rho-kinase In Inflammatory Cytokine Mediated Formation Of Coronary Artery Spasm And Tongxinluo Treatment In Pigs

Mechanism on the Expression ofRho - kinase in inflammatory cytokineMediated Formation of Coronaryartery spasmIntroductionAccording to epidemic research , CHD will become one of the key disease to the people lived in 21 centry, threatening people's life , hindering economic development. Phosphorylation of the myosin light chain ( MLC ) is one of the most important steps for vascular smooth muscle contraction. The level of MLC phosphorylation is determined by the balance between MLC phosphorylation by myosin light chain kinase(MLCK) and dephosphorylation by MLC phosphatase. Rho kinase is involved in the Ca2 + sensitivity of smooth muscle contraction. Rho kinase plays a key role in inducing vascular smooth hypercontration by inhibiting MLC phosphatase through the phosphorylation of MBS. This trial is to observe the mechanism on the expression of Rho - kinase in inflammatory cyto-kine mediated formation of coronary artery spasm.Materials and Methods1. Animal model: sixteen male yokshire pigs weighing 25 ~ 30 kg , aging 2 ~ 3 months are randomly divided into 2 groups: ( 1) control group;( 2 ) model group . After one week seeding, the animals were sedated with ketamine hydro-chloride and valin and anesthetized with sodium pentobarbital. The animals were then intubated and ventilated with room air. Oxygen was supplemented via a positive - pressure respirator. Under aseptic conditions, a left thoracotomy was performed. The proximal segments of LAD and LCX were gently wrapped with cot-ton mesh that had absorbed 0. 05ml of sepharose bead solution with or without recombinant human IL - 1(3. However, in the control group ,the selective coronary artery was wrapped without IL - lfj. The amount of IL - 1(3 is 2. 5u,g in model group2. Coronary angiography;Two weeks after the thoracotomy , the animals were anesthetized. Selective coronary angiography were performed to study ath-erosclerostic condition of coronary artery. The responses to the intracoronary administration of serotonin(10jxg/kg) were examined.3. Samples collection : After coronary arteriographic, the animals were killed with a lethal dose of sodium pentobarbital, the hearts were removed, the proximal segments of LAD, LCX were retain for histopathology and RT - PCR analyzing.4. The expression of Rho - kinase by Reversal Transcription Polymerase Chain Reaction(RT - PCR). The PCR products were subjected to 2% sepharose gel electrophoresis, and measured using an imaging analyzed .5. Measurement of Rho - kinase activity by western blot analysis;Myosin - binding subunit ( MBS) of myosin phosphatase, one of the substrates of Rho -kinase was measured by SDS - PAGE, followed by electrophoretic transfer ofthe proteins to a nitrocellulose membrane. The amounts of phosphorylated MBS were quantified by immunoblot procedures.6. Statistic Analysis: All the data were expressed as mean values standard deviation. Statistical significance was determined by t test.Result1. Result of coronary angiography;The coronary artery were free from stenosis in control group, slight stenosis in model group, intracoronary serotonin caused marked coronary vasoconstriction in model group, whereas the autacoids don't caused vasoconstriction in control group.2. Histology result;intimal thickening with newborn smooth muscel cells and many inflammatory cells infiltrated to the neointima and medial membrane were noted in model groups.3. Result of RT - PCR: The mRNA level of Rho - kinase were significantly greater in model group(63. 7% ±4.3 vs98.2% ±7.7,P <0.05).4. The result of western blot;The activity of Rho - kinase in the control group is siginificant greater than the control group (6510 ±779 vs 25485 ±4745, P<0.05).DiscussionAtherosclerosis is an inflammatory disease, the lesions of atherosclerosis rer present a series of highly specific cellular and molecular responses that can best be described as an inflammatory disease. Previous pathological studies have demonstrated an infiltration of inflammatory cells and mast cells in the adventitial surface of the coronary artery excised from patients with unstable angina and variant angina,respectively. It has been suggested from autopsy findings that adventitial mast cells may potentiate coronary atherosclerosis, vasospasm, and thrombosis in long - term cocaine abusers. It is therefore possible that adventitial inflammatory changes may be involved in the pathogenesis of coronary artery disease. Recently studies had suggest that inflammatory cytokines are associated with cardiovascular diseases. IL - 1 (3, one of the major inflammatory cytokines found in atherosclerosis, may have a wide variety of biological effects on cardiovascular systems. In our study ,we make a small amount of IL -1(3 absorbed to adventitia in order to obversve the relationship between adventitial inflammatory lesions and atherosclerosis and coronary spasm. Two weeks after the surgery , coronary angiography showed mild stenosis lesions were observed at the site treated with IL - 1 (3 beads, a histological examination reveals an intimal thickening at the IL - 1(3 - treated sites. Most of the cells in the thickened intima were smooth muscle cells and an accumulation of inflammatory mononuclear cells at the adventitial surfance and intimal was noted at the IL - 1 (3 - treated site. Simultaneous intracoronary serotonin caused coronary spasm at the IL - 1 (3 - treated segment,but not at the control site. The present study demonstrated that coronary inflammatory changes may be more important in the pathogenesis of atherosclerosis and coronary spasm.Several lines of evidence indicate that coronary artery spasm is caused primarily by smooth hypercontration. Phosphorylation of the myosin light chain (MLC) is one of the most important steps for vascular smooth muscle contraction, coroanry artery spasm is associated with an enhanced and sustained MLC monophosphorylation and the appearance of MLC diphosphorylation. Protein kinase C (PKC ) and Rho - kinase are substantially involved in the intracellular mechanism of the coronary spasm, resulting in increases in the mono - and diphosphorylation of myosin light chain (MLC). Rho - kinase has been identified as one of the effectors of the small GTP - binding protein Rho. In this stud-y, molecular biological analyses demonstrated that Rho - kinase is upregulated at the spastic site (at all levels, including mRN A, protein, and activity) compared with the control segment. These results indicate that Rho - kinase plays a important role in the in the pathogenesis of coronary spasm.The level of MLC phosphorylation is determined by the balance between MLC phosphorylation by myosin light chain kinase (MLCK) and dephosphoryla-tion by MLC phosphatase( MLCP) . Smooth muscle MLCP consist of a 38 - kDa catalytic subunit,the 130 - kDa MBS ,and a 21 - kDa subunit. MBS serves as a targeting subunit of MLCP to myosin and enhances the activity of the enzyme toward myosin. Study suggested that the generation of diphosphorylation MLC may be caused in part by inhibition of MLCP in smooth muscle cells, and also showed that treatment with calyculin A , an inhibitor of phosphatases, including MLCP, induced MLC diphosphorylations in smooth muscle cell without an increase in in-tracelluar Ca + levels by calcium ionophore does not result in an increase in diphosphorylated MLC. These lines of evidence suggest that inhibition of MLCP activity is essential for the occurrence of coronary artery spasm. In the present study , MBS phosphorylation at the IL - 1 (3 - treated site in response to serotonin was significantly increased, suggesting that MLCP activity is significantly suppressed in the spastic compared with the control segment, resulting in an increase in MLC phosphorylation on stimulation by serotonin. Recently it is reported that Rho - kinase phosphorylates MBS and reduces the MLCP activity in vitro. These findings suggest that MBS phosphorylation is mediated by Rho - kinase, resulting in inhibition of MLCP and subsequent increase in MLC phosphorylations. In ourporcine model, it is highly possible that upregulated Rho - kinase inhibits MLCP through MBS phosphorylation, and modulate Ca2 + sensization of vascular smooth muscle, resulting in the occurrence of coronary artery spasm.Conclusion1. chronic treatment with IL -13 induced coronary intimal lesions and selective vasospasmtic responses to autacoids in vivo.2 . coronary spasm was associated with an enhanced MBS phosphorylation that should have resulted in the inhibition of MLCP.3. Rho -kinase mediated this MBS phosphorylation,resulting in the occurrence of smooth muscle hypercontration.The secend part:The inhibition effect and action mechanismof TongXinLuo on IL -1 p - induced coronary artery spasm in small pigsIntroductionNow the main drugs of treatment in coronary artery spasm include calcium channel blockers and protein kinase inhibitor (fasudil). Tongxinluo capsule is a Chinese herbal medicine in the treatment of coronary artery disease, include coronary artery spasm. This trial is to observe the expression of Rho - kinase mR-NA during coronary artery spasm and to investigate the effects of Tongxinluo on the Rho - kinase and coronary artery spasm in swine model of coronary artery spasm induced by IL - 1 (3.Materials and Methods1. Preparation of animal model: The model of coronary coronary artery spasm in 16 male small pigs was established. After 2 weeks the state of coronary artery spasm induced by injecting 5 - hydroxytryptamine into coronary artery was observed, the 12 pigs with coronary artery spasm was randomly divided into 2 groups, the 6 pigs in control group were fed with special diet;the other 6 pigs in TongXinLuo group were fed with special diet and Tongxinluo (1 g/kg/d).2. Coronary angiography : After 4 week's feeding, Selective coronary angi-ography were performed . The responses to the intracoronary administration of serotonin(10|xg/kg) were examined.3. Samples collection :The same as the first part.4. The expression of Rho - kinase by Reversal Transcription Polymerase Chain Reaction ( RT - PGR ) : The same as the first part.5. Measurement of Rho - kinase activity by western blot analysis;The same as the first part.6. Statistic Analysis: All the data were expressed as mean values standard deviation. Statistical significance was determined by t test.Result1. Result of coronary angiography: The stenosis rate of coronary artery in control group are more obvious than in TongXinLuo group . intracoronary serotonin caused marked coronary vasoconstriction in control group, whereas the auta-coids don't caused vasoconstriction in TongXinLuo group.2. Histology result;intimal thickening with newborn smooth muscel cells and many inflammatory cells infiltrated to the neointima and medial membrane were noted in control groups. The intimal thickening were markedly inhibited by oral TongXinLuo treatment.3. Result of RT - PCR;The expression of mRNA of Rho - kinase were significantly greater in control group. As comparde with that of control group, The expression of Rho - kinase in TongXinLuo group was significantly inhibited (71. 5% ±2.4% vs98.2% ±7.7% ,P<0.05).4. The result of western blot;The activity of MBS - P in the control group is siginificant greater than TongXinLuo group. And this increased activity is inhibited by treatment with TongXinLuo(16633 ± 1390 vs 25485 ±4745, P <0.05).DiscussionCoronary artery spasm plays an important role in a wide variety of ischemic heart diseases, not only in variant angina but also in unstable angina , myocardial infarction ,and sudden death. Tongxinluo capsule is a Chinese herbal medicine in the treatment of heart disease. The composition of Tongxinluo capsule includeRadix Ginseng, Scorpio, Hirudo, Eupolyphaga seu Steleophage, Scolopendra, Periostracum Cicadae, Radix Paeoniae Rubra, and Borneolum Syntheticum. Pharmacological studies showed that the complex drug could improve the myocardial ischemia myocardial infarction significantly and prevent the occurrence of arrhythmia and hyperlipidemia obviously, increase the coronary arterial blood flow, dilate the coronary vessels, improve myocardial blood supply. Our study show that TongXinLuo could obviously inhibit coronary artery spasm induced by 5 -hydroxytrptamine .Rho kinase has been identified as one of the effectors of the small GTP -binding protein Rho. Accumulating evidence has demonstrated that the Rho kinase/Rho mediated pathway plays an important role in coronary artery spasm. Rho - kinase phosphorylates MBS and result in inhibition of MLCP and a subsequent increase in MLC phosphorylations.Our study find that the 10(83% ) vascular section in the Tongxinluo group were negative in vascular section for 5 - hydroxytryptamine - induced spasm, 2 (17% ) vascular section was positive, the 11(91.7%) vascular section in the control group were positive in vascular section for 5 - hydroxytryptamine - induced spasm, 1(8. 3% ) vascular section was negative. The expression of mRNA of Rho kinase and the phosphorylation levels in control group were increased. So, TongXinLuo could obviously inhibit coronary artery spasm induced by 5 — hydroxytrptamine by inhibiting the expression of mRNA of Rho kinase and the decrease the levels of MBS - P.Conclusion1. Chronic treatment with IL — 13 induced coronary intimal lesions and selective vasospasmtic responses to autacoids, and the efffect can be inhibit by TongXinLuo in vivo.2. Rho kinase is involve in IL - lfjmediated formation of coronary spasm in pig in vivo, and the increase of Rho kinase in coronary spasm in pig can inhibit by TongXinLuo.. 3. TongXinLuo can inhibit the expression of MBS phosphorylation.4. TongXinLuo could obviously inhibit coronary artery spasm induced by 5- hydroxytrptamine by inhibiting the expression of mRNA of Rho kinase and the decrease the levels of MBS - P.