The Mechanism Of Ketanserin In Preventing Endotoxic Shock Via Inhibition Of INOS

Backgrounds and Objectives: Sepsis is a systemic inflammatory response to thepresence of infection, mediated via the production of many cytokines, including tumournecrosis factor α (TNF-α), interleukin (IL)-6, and IL-1, as well as nitric oxide (NO) whichcauses changes in the circulation, increases endothelial permeability and actives thecoagulation pathways. These mechanisms play roles during septic shock, but on a systemicscale, leading to diffuse endothelial disruptior vascular permeability, vasodilation, andthromnosis of end-organ capillaries. Endothelial damage itself can further activateinflammatory and coagulation cascades, creating a positive feedback loop, and leading tofurther endothelial and end-organ damage. Treatment of septic shock is based on support oforgan perfusion with fluids, administering vasoactive agents and antibiotics. Although, thepathophysiology of septic shock and the process of inflammation are understand moredeeply, the mortality rates are16%with sepsis,20%with severe sepsis, and46%withseptic shock. Therefore, it is important to find new strategies for septic shock treatment andaccurate mechanism.Ketanserin, an antihypertensive drug with a central sympathoinhitory action, is aselective5-HT2Areceptor antagonist with additional α1-adrenoceptor-blocking prooerties.Studies have demonstrated that ketanserin effectively lowers blood pressure (BP),decreases blood pressure variability (BPV) and enhances BRS in SHR. However, clinicalapplication of ketanserin was not limited to anti-hypertension. Research demonstrated thatintracoronary administration of ketanserin augments coronary collateral flow and decreasesmyocardial ischemia during balloon angioplasty. This could be of clinical significance inthe management of acute ischemic syndromes. Another clinical research showed thattreated with2%ketanserin ointment for8weeks, observed a significant decrease inrelative wound area compared with the placebo group in chronic leg ulcers patients. Theseresults indicate that ketanserin is a valuable therapy for difficult-to-treat leg ulcers. Thiseffect of ketanserin is through blocked of5-HT2Areceptor and improved microcirculation.Our previous study demonstrated that ketanserin could protect the organ damage in hypertension by restoring arterial baroreflex function. Furthermore, we had demonstratedthat ketanserin has a beneficial effect in septic shock through restoration of baroreflexfunction.Nitric oxide (NO) is one of many vasoactive substances released, from a variety ofcells, under conditions of endotoxaemia and sepsis. Under physiological conditions it isproduced by two constitutive calcium-dependent enzymes (nitric oxide synthase; NOS) inneurons (nNOS) and endothelial cells (eNOS) and has functions ranging fromneurotransmission and vasodilatation to inhibition of platelet adhesion and aggregation.Following bacterial infection, especially with Gram-negative organisms, the formation ofNO from L-arginine is enhanced due to the induction of a NOS enzyme (iNOS) in cardiacmyocytes and vascular smooth muscle cells. Studies ascertained that the excess NO formedby iNOS resulted in the pathophysiology of sepsis shock, including haemorrhage,vasodilation, intravascular coagulation, and hypotension.When infection occurs, many signaling molecules are activated, such as nuclearfactor-κB (NF-κB), cyclic AMP responsive element-binding protein (CREB) andmitogen-activated protein kinases (MAPKs) family, which are classified into at least threecomponents: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK),and p38MAPK. Recaent studies have implicated that NF-κB and MAPKs induce therelease of immune-related cytotoxic factors such as iNOS, COX-2, and proinflammatorycytokines.Considering the critical role of iNOS in the sepsis shock, we hypothesized thatketanserin cures the sepsis shock via inhibiting the expression of iNOS. Furthermore, weexplained the molecular mechanism.Methods: The study was based on the model of endotoxin-induced shock mice andRAW264.7stimulated by LPS. Kunming mice were injected with LPS (30mg/kg, i.p.) toinduce septic shock. Ketanserin was administered (3mg/kg,10mg/kg, i.p.) immediatelyafter injuected with LPS for12hours. The expression of iNOS was detected by Westernblot and immunohistochemistry. The level of NO was measured by Griess reagent. RAW264.7cells were pre-treated with indicated ketanserin for10minutes, then stimulatedwith LPS (100ng/ml) for indicated time. Then, the expression of iNOS protein andMAPKs were detected using Western blot. The level of iNOS mRNA was measured byreal-time PCR. The cells were treated with different dosage of ritanserin or prazosin for10minutes and stimulated with LPS (100ng/ml) for8hours in order to measure the role onthe expression of iNOS. Moreover, the activity of NF-κB was measured using EMSA.Results：1. Kunming mice were injected with LPS (30mg/kg, i.p.) to induce septic shock,then injected ketanserin (3mg/kg,10mg/kg, i.p.) immediately after injected withLPS for12hours. Results showed that ketanserin could both inhibited theexpression of iNOS protein and decreased the level of NO, especially the dosge of10mg/kg.2. RAW264.7cells were pre-treated with ketanserin (10μM) for10min andstimulated with LPS (100ng/ml) for indicated time (0-12h).The expression ofiNOS was inhibited at the early time period of the iNOS induced, especially at8hours after stimulated with LPS. Furthermore, RAW264.7cells were pre-treatedwith ketanserin (0.1-10μM) and stimulated with LPS (100ng/ml) for8hours.Results show that ketanserin could inhibit the expression of iNOS and the level ofNO dose-inpendently3. RAW264.7cells were pre-treated with ketanserin (1μM,10μM) for10min andstimulated with LPS (100ng/ml) for5hours. The level of iNOS mRNA wasdecreased by ketanserin significantly.4. RAW264.7cells were pre-treated with ketanserin (0.1-10μM) for10minutes andstimulated with LPS (100ng/ml) for indicated time. The level TNF-α and IL-6were significantly decreased by ketanserin.5. RAW264.7cells were pre-incubated with ritanserin (0-10μM) or prazosin(0-1μM) for10minutes, then stimulated with LPS (100ng/ml) for8hours. Theexpression of iNOS was examined by western blotting. Ritanserin, a specific antagonist of5-HT2A receptor, could significantly inhibit the expression of iNOSin RAW264.7cells stimulated with LPS. However, prazosin which is a specificantagonist of α1receptor, failed to inhibit the expression of iNOS.6. RAW264.7cells were pre-treated with ketanserin (10μM) for10min andstimulated with LPS (100ng/ml) for indicated time (0-2hours). The effect ofketanserin on the phosphorylation of MAPKs was detected by Western blot. Theresults showed ketanserin could inhibit the phosphorylation of ERK1/2, while hadno effect on that of p38and JNK. Moreover, RAW264.7cells were pre-treatedwith ketanserin (10μM) or PD98059(40μM) for10min and stimulated withLPS (100ng/ml) for8h. The effect of ketanserin on iNOS was canceled.7. RAW264.7cells were pre-treated with ketanserin (10μM,100μM) for10minutes and stimulated with LPS (100ng/ml) for8hours. Result showed that theactivity of NF-κB was inhibited by ketanserin.Conclusion: Ketanserin could bind with5-HT2Areceptor and inhibit iNOS pathwaythrough ERK1/2signaling pathway.