| Objective To investgate the effects of anandamide on the proliferation and death of primaryHSC isolated from SSLF mice.Methods1) Making liver injury mice models of different stages by applying cercaria to theskin of mice, and identified by HE and Masson dying, the indexes of liver、spleen andskindey were analyzed.2) ALT and AST were quantified by automatic biochemical analyzer.3) HSCs were isolated by the discontinuous density gradient centrifugationtechnique and identified by double dying of α-SMAand desmin, the cell viability wasmeasured by Trypan Blue exclusion assay.4) MTT assay was used to test the effect of AEA on HSCs proliferation,IC50wascalculated by SPSS software.5) HSCs death induced by AEA was analyzd by Annexin V-FITC/PI flowcytometry.6) The morphological change of HSC nucleus was observed by DAPI dying.Results1)HE and Masson dying showed that in the6weeks post-infect mice livers, roundgranulomatous was formed, eggs deposited in the blood vessel, inflammatory cellsdiffusated, blue and round collage fibers deposited.With the time development, the degreeof hepatic cells necrosis and liver fibrosis aggravated.Compared to the liver and spleenindexes(4.75%±0.80%,0.43%±0.07%)of normal mice, those of6,9,11weeks post-infectmice enhanced(liver index:6.27%±0.56%,8.40%±1.43%,7.48%±1.32%,P＜0.05,P＜0.01;spleen index:1.24%±0.37%,2.47%±0.46%,2.62%±1.23%,P＜0.05,P＜0.01),howeverthe skindey index had no change.2)Comparing with those of normal mice(ALT:30.90±1.44,AST:114.00±5.07),ALT and AST become to increase from3weeks post-infect（ALT:91.00±1.36,AST: 232.10±2.54,P＜0.01), and reach the peak in6weeks(ALT:176.22±7.08,AST:319.67±9.96，P＜0.01), however decrease in9（ALT：103.60±4.34，AST：197.40±5.58，P＜0.01)and11weeks（ALT：50.13±3.12，AST：203.88±4.14，P＜0.01）,but still up tothe normal levels.3) The purity of isolated primary HSCs was95%,cell viability was90%.4) The inhibition rates of different concentration AEA in primary HSCs were asfollows:5.14%±1.61%at5μM、10.34%±3.22%at10μM、14.12%±3.98%at20μM、18.64%±3.67%at40μM、23.86%±3.06%at60μM、62.81%±7.75%at80μM、70.39%±7.11%at100μM、72.31%±6.71%at120μM. And IC50value of AEA was79.06±9.38μM.5) The death rates of different concentration AEA in primary HSCs were9.5%±3.19%at0μM、9.8%±2.28%at5μM、11.18%±1.82%at5μM、12.2%±2.39%at20μM、15.1%±1.88%at40μM、45.85%±4.73%at60μM、63.04%±7.00%at80μM、73.32%±4.62%at100μM、89.47%±8.84%at120μM.6) The HSC nucleus treated by AEA was cracked,chromation were condensed,the contents of some cells were overflowed out of the nucleus.Conclusions After6weeks post-infect, granulomatous and fibrosis are formed in the livers,the livers and spleen become enlargement, applying cercaria to the skin of mice can buildacute and chronic schistosoma liver injury models.AEA can inhibit primary HSCsproliferation and promoted death in a concentration-dependent manner. Objective To explore the roles of lipid raft, cannabinoids receptor and oxidative stress inthe effects of AEA-induced primary HSCs proliferation and death, MAPK signalingpathways, and the expressions of cytokines IL-6,MCP-1,TNF-α,IL-1β.Methods1) The relations of time and concentratin between AEA and MAPK signalingpathway family members P-Erk1/2,P-P38and P-JNK1/2were tested by Western-blotting.2) Lipid raft was destroyed by MCD, SR141716and AM630were used to blockAEA to combine with respective receptors CB1and CB2, NAC was to clean oxygen freeradical, the cell death induced by AEA combination with the above managements wasobserved by Annexin V-FITC/PI flow cytometry.3）MTT was used to observe the roles of MCD,SR141716,AM630and NAC in theAEA induced primary HSC proliferation inhibition.4) MAPK signaling pathways activation and caspase-3expression were observedby Western-blotting before and after the lipid rafts were obstructed, the block to combinewith the receptors and oxygen free radical production.5) The effects of AEA on the expressions of cytokines IL-6,MCP-1,TNF-α, IL-1βwere observed by RT-PCR before and after the lipid rafts were obstructed, the block tocombine with the receptors and oxygen free radical production.Results1) AEA promoted the expression of P-Erk1/2,P-P38and P-JNK1/2in a time andconcentration dependent manner: AEA started to promote the expression of P-Erk1/2,P-P38and P-JNK1/2at20μM, and increased with the concentration enhancement（all P＜0.05）.The expressions of P-Erk1/2and P-P38increased obviously at the time of 15min,30min,1h,3h,6h, and P-JNK1/2was at15min,30min,1h, after that the expressiondecrease.2) The cell death rates induced by AEA decreased after combination with MCDand NAC(both P＜0.05), there were no changes when combined with SR141716andAM630.3)MCD and NAC reduced AEA induced primary HSCs proliferation inhibition(P＜0.05), not SR141716andAM630.4) The expression of P-Erk1/2,P-P38and P-JNK1/2induced by AEA decreasedwhen combinated with MCD, SR141716and NAC（all P＜0.05）, however AM630onlyreduced the expression of P-P38（P＜0.05）, none of AEA, MCD, SR141716, AM630andNAC affected the expression of caspase-3.5) AEA enhanced the expression of IL-6and reversed by MCD, SR141716, AM630and NAC（all P＜0.05）.The inhibition of MCP-1by AEA was reversed by AM630（P＜0.05）,but MCD, SR141716and NAC had no effect on the inhibition. The reduction ofTNF-α and IL-1β induced by AEA was further reduced by MCD, SR141716and NAC（allP＜0.05）,however reversed by AM630（P＜0.05）.Conclusions AEA can promote primary HSC necrosis through activating MAPK signalingpathways in a time and concentration manner. Lipid raft and ROS were necessary for thecell necrosis and proliferation inhibition induced by AEA, independent of CB.AEA may bethe immunal molecule which regulates the expression of cytokines. Objective To observe the effects of different concentration AEA on ROS production, andthe expression of NADPH oxdase subunits gp91-phox and p47-phox when combinationwith MCD.To explore the roles of lipid raft, CB and NADPH oxdase in the ROSproduction induced by AEA in primary HSC.Methods1) DCFH-DA could freely enter HSC through cytomembrane, and hydrolyzedinto DCFH by esterases, DCFH could not pass through cytomembrane, and the probeDCFH was carried to HSCs. Reactive oxygen could oxidize non-fluorescent DCFH intofluorescent DCF, the intensity of DCF tested by flow cytometry indirectly reflect the levelof ROS.2) Western-blotting was used to observe the effects of MCD on the AEA-inducedNADPH oxdase subunits gp91-phox and p47-phox expressions, and the rates of respectivestripe mean gray value to β-actin were analyzed to quantify the protein.3) DCF flow cytometry to detect the effects of MCD, SR141716and AM630, theNADPH oxdase activity inhibitor DPI and Apocynin, antioxidant NAC on AEA-inducedROS in primary HSC.Results1)The level of ROS increase with the concentration enhancement.The ROS levelsof different concentration AEA were:4.4±1.52at0μM,24.52±7.35at5μM,28.31±5.87at10μM,47.07±7.92at20μM,61.22±10.69at40μM,89.99±16.35at60μM,106.48±19.58at80μM,95.93±13.74at100μM,96.78±14.83at120μM。2)AEA could promote the expression of NADPH oxdase subunits gp91-phox andp47-phox（both P＜0.05）, and decreased when combinated with MCD（P＜0.05）.①gp91-phox/β-actin：control group0.13±0.01,20μM AEA group0.29±0.03,60μM AEAgroup0.35±0.02,60μM AEA+MCD group0.20±0.01.②p47-phox/β-actin:control group0.06±0.01,20μM AEA group0.40±0.04,60μM AEA group0.66±0.06，60μM AEA+MCD group0.36±0.03。3)MCD and NAC reduced AEA-induced ROS production（both P＜0.05）,NADPHoxdase activity inhibitors DPI and Apocunin could partly decrease AEA-induced ROSproduction,however the statistical difference was no significant（P＞0.05）.The ROS levelsof respective groups were DMSO group6.52±2.78，20μM AEA group48.75±6.81,60μMAEA group105.87±15.79,60μM AEA+SR141716group107.85±10.57,60μMAEA+AM630group108.57±19.38,60μM AEA+MCD group40.35±5.82,60μMAEA+DPI+Apocynin group94.39±21.94,60μM AEA+MCD group35.96±7.59.Conclusions①AEA could promote ROS production in a concentration-dependent mannerin primary HSC.②The expressions ofNADPH oxdase subunits gp91-phox and p47-phoxinduced by AEA are lipid raft-dependent.③AEA promotes primary HSC ROS productionby activating NADPH oxdase subunits gp91-phox and p47-phox, however inhibition ofNADPH oxdase activity could not prevent the AEA induced ROS production, whichsuggests NADPH oxdase is not the main source of ROS induced by AEA. Objective To observe the effects of anandmide on liver function and pathological changesof SSLF mice.Methods1) SSLF mice models were prepared, different dose AEA （5mg/kg and10mg/kg）was respectively injected into SSLF mice, once every day for4weeks, theindexes of liver and spleen were analyzed.2) ALT and AST were tested by automatic biochemical analyzer.3) The psthological changes of various groups were observed by HE dying.4) The deposition of collagen fibers was observed by Masson dyming, and thequantification was analyzed by Image-pro-plus software.Results1) The SSLF mice models were successfully prepared. Comparing with those ofnormal mice, the indexes of liver and spleen in model group、5mg/kg AEA group and10mg/kg AEA group obviously increased （all P＜0.05）.However when compared withmodel group, the indexes of liver and spleen in5mg/kg AEA group and10mg/kg AEAgroup had no evident changes.2)Comparing with those of normal mice,ALT and AST levels of model group、5mg/kg AEA group and10mg/kg AEA group obviously raised（all P＜0.05）. Whencompared with model group, both of ALT and AST of the group treated with5mg/kg and10mg/kg AEA increased（both P＜0.05）,however there was no statistical significancebetween5mg/kg AEA group and10mg/kg AEA group.3)In the livers of model group、5mg/kg AEA group and10mg/kg AEA group mice,HE dying showed that eggs deposited in the blood vessel, actue and chronic granulomatousformed, inflammatory cells diffusation.Compared with that of model group, the area ofgranulomatous and inflammatory cells of5mg/kg and10mg/kg AEA treated mice diffusation increased.4)Masson dying showed that except those consisted of blood vessel, there were nocollagen fibers deposition, however collagen fibers deposited around eggs in the livers ofmodel group and AEA treated group mice, and the quantification of collagen fibersincreased（all P＜0.05）.Comparing with that of model mice, neither of collagen fibers inthe livers of5mg/kg AEA group and10mg/kg AEA group had statistical significance.Conclusions Short-term AEA application could aggravate the inflammation of SSLF mice,but had no effect on preventing liver fibrosis proceeding. Objective To observe the expressions of MMP-9, TIMP-1, α-SMA and activation ofMAPK/AKT signaling pathways induced by S. japonicum egg antigen in primary HSCs.Methods1) At the RNA level, the expressions of MMP-9, TIMP-1, α-SMA induced bysoluble egg antigen (SEA) at different concentration were observed by RT-PCR.2) At the protein level, the expressions of MMP-9, TIMP-1, α-SMA andactivation of P-P38,P-JNK and P-AKT induced by soluble egg antigen at differentconcentration were detected by Western-blotting.Results1) At the RNA level, SEA at50μg/ml and250μg/ml promoted the mRNAexpression of α-SMA and TIMP-1（all P＜0.05）,and reduced the expression of MMP-9（allP＜0.05）.2) At the protein level, SEA at50μg/ml and250μg/ml promoted α-SMA andinhibited MMP-9protein expression (all P＜0.05),however SEA had no effect on theprotein expression of TIMP-1.3) At the protein level, all of concentrations from5μg/ml to250μg/ml of SEApromoted P-P38activation（both P＜0.05）, the concentrations of50μg/ml and250μg/mlwere obvious. SEA at15μg/ml,50μg/ml and250μg/ml promoted P-JNK1/2expression (allP＜0.05), only at250μg/ml could promote P-AKT activation.Conclusions S.japonicum SEA can promote liver fibrosis through P38/JNK MAPKsignaling pathways activation at earlier stages, and P38/JNK MAPK＆AKT signalingpathways at later stages. |
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