内源性大麻素anandamide和虫卵抗原对血吸虫肝纤维化小鼠体内/外影响的初步探讨
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摘要
目的研究内源性大麻素AEA对血吸虫肝纤维化小鼠原代肝星状细胞（HSC）增殖及细胞死亡的影响。
方法1）应用尾蚴贴敷腹部皮肤的方法建立不同时期的血吸虫肝损伤小鼠模型，肝脏组织的HE染色及Masson染色证实模型建立成功，分别计算肝脏组织、脾脏组织和肾脏组织的指数。
2）全自动生化仪检测血吸虫肝纤维化小鼠模型血清中的转氨酶（ALT和AST）含量。
3）应用非连续梯度密度离心法分离原代HSC，α-SMA和desmin免疫荧光双重染色鉴定分离的细胞，胎盘蓝排斥实验计算分离的细胞的活性。
4）应用噻唑蓝（MTT）比色法检测AEA对原代HSC增殖的影响并用SPSS软件计算AEA的IC50。
5）应用Annexin V-FITC/PI双标的流式细胞术检测AEA诱导的HSC死亡。
6）应用DAPI染色观察在AEA的作用下原代HSC细胞核形态的变化。
结果1）HE染色和Masson染色显示，在尾蚴感染的第6周小鼠的肝脏组织出现类圆形的嗜酸性肉芽肿，虫卵在血管沉积，炎症细胞浸润，蓝色的胶原纤维在肝脏组织呈同心圆状沉积。随着时间的推移，肝细胞的嗜酸性坏死和纤维化程度逐渐加重。与正常组的肝脏指数和脾脏指数(4.75%±0.80%,0.43%±0.07%)比较，第6、9、11周尾蚴感染后小鼠肝脏指数（6.27%±0.56%，8.40%±1.43%，7.48%±1.32%，P＜0.05，P＜0.01）和脾脏指数（1.24%±0.37%，2.47%±0.46%，2.62%±1.23%，P＜0.05，P＜0.01）明显升高，而肾脏指数则没有明显变化。
2）与正常组（ALT：30.90±1.44，AST：114.00±5.07）相比，谷丙转氨酶（ALT）和谷草转氨酶（AST）在第3周开始明显升高（ALT：91.00±1.36，AST:232.10±2.54，P＜0.01），第6周达到高峰（ALT：176.22±7.08，AST:319.67±9.96，P＜0.01），在第9周（ALT：103.60±4.34，AST：197.40±5.58，P＜0.01）和第11周（ALT：50.13±3.12，AST：203.88±4.14，P＜0.01）下降但仍明显高于正常水平。
3）α-SMA和desmin免疫荧光双重染色发现，分离的HSC纯度为95%，细胞活性为90%。
4）MTT比色法显示不同浓度的AEA对原代HSC增殖的抑制率分别为：5μM组为5.14%±1.61%、10μM组为10.34%±3.22%、20μM组为14.12%±3.98%、40μM组为18.64%±3.67%、60μM组为23.86%±3.06%、80μM组为62.81%±7.75%、100μM组为70.39%±7.11%、120μM组为72.31%±6.71%，AEA的IC50值为79.06±9.38μM。
5）Annexin V-FITC/PI双标的流式细胞术的结果显示，不同浓度的AEA诱导原代HSC的死亡率分别为：0μM组为9.5%±3.19%、5μM组为9.8%±2.28%、10μM组为11.18%±1.82%、20μM组为12.2%±2.39%、40μM组为15.1%±1.88%、60μM组为45.85%±4.73%、80μM组为63.04%±7.00%、100μM组为73.32%±4.62%、120μM组为89.47%±8.84%。
6）DAPI染色的结果显示AEA处理的原代HSC的细胞核肿胀破裂，染色质固缩成颗粒状，部分细胞的核内容物溢出。
结论在尾蚴感染后的第6周，小鼠的肝脏组织出现嗜酸性肉芽肿和胶原纤维及虫卵的沉积，肝脏组织和脾脏组织逐渐肿大，尾蚴贴敷腹部皮肤的方法可成功建立早期及晚期血吸虫肝损伤小鼠模型。AEA可呈浓度依赖性地抑制原代HSC的增殖并促进其坏死。
目的探讨原代HSC胞膜的脂筏、大麻素受体(CB)及氧化应激对AEA诱导的原代HSC增殖坏死及MAPK信号通路的影响，对细胞因子IL-6，MCP-1， TNF-α及IL-1β表达的影响。
方法1)Western-blotting检测AEA与MAPK主要成员P-Erk1/2,P-P38和P-JNK1/2的时间及浓度的关系。
2)分别用胆固醇耗竭剂MCD破坏脂筏的完整性，CB1受体拮抗剂SR141716及CB2受体拮抗剂AM631阻断AEA与其相应受体的结合，抗氧化剂NAC清除自由基，应用Annexin V-FITC/PI双标的流式细胞术观察上述处理对AEA诱导的原代肝星状细胞死亡影响。
3)应用MTT观察MCD，SR141716和AM639及NAC对AEA诱导的原代HSC死亡率的影响。
4)Western-blotting观察MCD，SR141716和AM639及NAC对AEA诱导的MAPK信号通路激活及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)表达的影响。
5)RT-PCR检测MCD，SR141716和AM639及NAC对AEA诱导的细胞因子IL-6，MCP-1，TNF-α及IL-1β表达的影响。
结果1)AEA可呈时间和浓度依赖性地促进P-Erk1/2,P-P38及P-JNK1/2的表达：在AEA浓度为20μM的时候开始促进P-Erk1/2,P-P38及P-JNK1/2的表达，随浓度升高表达逐渐增加（P均＜0.05）。在AEA分别孵育15min,30min,1h,3h,6h时P-Erk1/2和P-P38的表达较高，而后逐渐下降。在15min,30min,1h时，P-JNK1/2的表达较高，而后逐渐下降。
2)MCD和NAC可降低AEA诱导的原代HSC的死亡率（P均＜0.05），而SR141716和AM630则无明显作用。
3)MCD和NAC可降低AEA对原代HSC增殖的抑制（P＜0.05），而大麻素受体则无明显作用。
4)MCD、SR141716和NAC可抑制AEA诱导的P-Erk1/2、P-P38及P-JNK1/2的表达（P均＜0.05），AM630仅能抑制P-P38的表达（P＜0.05），而上述处理因素均对原代HSC内caspase-3的表达无明显影响。
5)AEA能诱导IL-6的表达增加，MCD、SR141716、AM630及NAC均可降低AEA诱导的IL-6的表达（P均＜0.05）。AEA可抑制MCP-1的表达（P＜0.05），MCD、SR141716及NAC对AEA诱导的MCP-1表达则无明显的影响，而AM630则能明显促进MCP-1的表达（P＜0.05）。AEA能降低TNF-α和IL-1β的表达（P均＜0.05），MCD、SR141716及NAC可进一步抑制AEA诱导的TNF-α和IL-1β的表达（P均＜0.05），而AM630则增强AEA诱导的TNF-α和IL-1β的表达（P均＜0.05）。结论内源性大麻素AEA可通过呈浓度和时间依赖性地激活MAPK信号通路促进原代HSC的坏死。脂筏及氧化应激是AEA诱导原代HSC坏死和抑制其增殖的必要条件，而与其胞膜的特异性大麻素受体无关。AEA可能是体内调节细胞因子表达的免疫分子。
目的观察不同剂量的内源性大麻素AEA对原代HSC内ROS的影响，观察AEA和MCD对NADPH氧化酶亚基gp91-phox和p47-phox表达的影响，并观察脂筏、大麻素受体及NADPH氧化酶在AEA诱导原代HSC内ROS产生过程中的作用，
方法1）利用荧光探针DCFH-DA可自由进入HSC胞膜，细胞内的酯酶水解生成DCFH。而DCFH不能通透细胞膜，从而使探针被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。在流式细胞仪上检测DCF的强度可间接反映原代HSC内ROS的水平。
2）Western-blotting检测MCD对AEA诱导的原代HSC内NADPH氧化酶亚基gp91-phox和p47-phox表达的影响，计算各组目的条带与β-actin灰度值的平均比值反应蛋白表达量。
3）观察大麻素受体拮抗剂SR141716或AM630，MCD及NADPH氧化酶抑制剂DPI和Apocynin，抗氧化剂NAC对AEA诱导的原代HSC内ROS产生的影响。
结果1）随着内源性大麻素AEA浓度的升高，原代HSC内ROS的水平逐渐升高。不同浓度的AEA诱导的ROS水平分别为：0μM组4.4±1.52，5μM组24.52±7.35，10μM组28.31±5.87，20μM组47.07±7.92，40μM组61.22±10.69，60μM组89.99±16.35，80μM组106.48±19.58，100μM组95.93±13.74，120μM组96.78±14.83。
2）AEA可促进原代HSC内NADPH氧化酶亚基gp91-phox和p47-phox的表达（P均＜0.05），加入MCD后其表达明显降低（P＜0.05）。①gp91-phox：各组与β-actin的平均比值分别为阴性对照组0.13±0.01，20μM AEA组0.29±0.03，60μM AEA组0.35±0.02，60μM AEA+MCD组0.20±0.01。②p47-phox：各组与β-actin的平均比值分别为阴性对照组0.06±0.01，20μM AEA组0.40±0.04，60μM AEA组0.66±0.06，60μMAEA+MCD组0.36±0.03。
3）SR141716和AM630并不能阻止AEA诱导的原代HSC内ROS的产生，而加入MCD和NAC后AEA诱导的原代HSC内ROS的水平明显降低（P均＜0.05），加入NADPH氧化酶抑制剂DPI和Apocunin后AEA诱导的原代HSC内ROS产生虽然有所降低，但差异没有统计学意义（P＞0.05）。各组的ROS水平分别为DMSO组6.52±2.78，20μMAEA组48.75±6.81，60μMAEA组105.87±15.79，60μMAEA+SR141716组107.85±10.57，60μMAEA+AM630组108.57±19.38，60μMAEA+MCD组40.35±5.82，60μMAEA+DPI+Apocynin组94.39±21.94，60μM AEA+MCD组35.96±7.59。
结论①AEA可呈浓度依赖性地促进原代HSC内ROS的产生。②内源性大麻素AEA促进NADPH氧化酶亚基gp91-phox和p47-phox的磷酸化依赖原代HSC胞膜脂筏的完整性。③AEA通过促进NADPH氧化酶亚基的磷酸化激活该酶，促进原代HSC内ROS的产生，而抑制NADPH氧化酶的活性后并不能阻止AEA诱导的ROS产生，表明NADPH氧化酶并不是AEA诱导原代HSC产生的ROS的主要来源。
目的观察AEA对血吸虫肝纤维化小鼠肝功能和病理学的影响。
方法1）制备血吸虫肝纤维化小鼠模型，将不同剂量的AEA（5mg/kg和10mg/kg）分别腹腔注射入小鼠体内，1次/天，共4周，计算各组每只小鼠的肝脏指数、脾脏指数。
2）全自动生化仪检测各组小鼠血清内转氨酶ALT和AST的含量。
3）HE染色观察各组肝脏组织的病理学变化。
4）Masson染色观察胶原纤维的沉积情况，并用Image-pro-plus软件计算胶原纤维的含量。结果1）血吸虫肝纤维化小鼠模型制备成功。与正常小鼠相比，模型组、5mg/kgAEA组和10mg/kgAEA组的肝脏指数、脾脏指数明显升高（P均＜0.05）。与模型组比较，5mg/kg AEA组和10mg/kg AEA组的肝脏指数和脾脏指数则无明显的变化。
2）与正常组比较，模型组、5mg/kg AEA组和10mg/kg AEA组的ALT和AST的含量明显升高（P均＜0.05）。与模型组比较，5mg/kgAEA组和10mg/kgAEA组的ALT和AST明显升高，差别具有统计学意义（P均＜0.05），而5mg/kg AEA组和10mg/kgAEA组在ALT和AST则无明显统计学意义。
3）HE染色结果显示与正常小鼠的肝脏组织相比，模型组、5mg/kg AEA组和10mg/kg AEA组均出现虫卵在血管的沉积，急慢性嗜酸性肉芽肿的形成，炎症细胞的浸润。与模型组比较，5mg/kgAEA组和10mg/kgAEA组的急慢性嗜酸性肉芽肿结节和炎症细胞浸润的面积增加。
4）Masson染色显示，在正常小鼠的肝脏组织中除组成血管的胶原纤维外，无胶原纤维的沉积，在模型组、5mg/kgAEA组和10mg/kgAEA组出现明显的围绕血管呈同心圆状的胶原沉积，胶原纤维的含量明显增加（P均＜0.05），而与模型组相比，5mg/kg AEA组和10mg/kg AEA组胶原纤维的含量无明显的统计学意义。
结论短期应用AEA可加剧血吸虫肝纤维化模型小鼠的肝功能，不能阻止肝纤维化的发展。
目的观察日本血吸虫可溶性抗原对原代HSC纤维化指标MMP-9、TIMP-1和α-SMA及P38/JNK MAPK信号通路和AKT信号通路的影响。
方法1）荧光定量RT-PCR检测RNA水平不同剂量的可溶性血吸虫抗原对MMP-9、TIMP-1和α-SMA表达的影响。
2）Western-blotting检测蛋白水平不同剂量的可溶性血吸虫抗原对MMP-9、TIMP-1和α-SMA表达及P38/JNK MAPK信号通路和AKT信号通路的影响。结果1）在RNA水平，与阴性对照相比，可溶性血吸虫抗原在50μg/ml和250μg/ml时可明显促进α-SMA和TIMP-1的表达（P均＜0.05），降低MMP-9的表达（P均＜0.05）。
2）在蛋白水平，与阴性对照相比，可溶性血吸虫抗原在50μg/ml和250μg/ml时可明显促进α-SMA和抑制MMP-9的表达（P均＜0.05），而对TIMP-1的表达没有明显的影响。
3）在蛋白水平，可溶性血吸虫抗原从5μg/ml至250μg/ml均可促进P-P38的表达，以50μg/ml和250μg/ml较为明显（P均＜0.05），在15μg/ml、50μg/ml和250μg/ml时促进P-JNK1/2的表达（P均＜0.05），而只有在250μg/ml时才促进P-AKT的表达（P＜0.05）。
结论日本血吸虫可溶性抗原在肝纤维化的早期和晚期通过P38/JNK MAPK信号通路促进其发生和发展，在晚期则可同时激活AKT信号通路促进其发展。
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.
方法1）应用尾蚴贴敷腹部皮肤的方法建立不同时期的血吸虫肝损伤小鼠模型，肝脏组织的HE染色及Masson染色证实模型建立成功，分别计算肝脏组织、脾脏组织和肾脏组织的指数。
2）全自动生化仪检测血吸虫肝纤维化小鼠模型血清中的转氨酶（ALT和AST）含量。
3）应用非连续梯度密度离心法分离原代HSC，α-SMA和desmin免疫荧光双重染色鉴定分离的细胞，胎盘蓝排斥实验计算分离的细胞的活性。
4）应用噻唑蓝（MTT）比色法检测AEA对原代HSC增殖的影响并用SPSS软件计算AEA的IC50。
5）应用Annexin V-FITC/PI双标的流式细胞术检测AEA诱导的HSC死亡。
6）应用DAPI染色观察在AEA的作用下原代HSC细胞核形态的变化。
结果1）HE染色和Masson染色显示，在尾蚴感染的第6周小鼠的肝脏组织出现类圆形的嗜酸性肉芽肿，虫卵在血管沉积，炎症细胞浸润，蓝色的胶原纤维在肝脏组织呈同心圆状沉积。随着时间的推移，肝细胞的嗜酸性坏死和纤维化程度逐渐加重。与正常组的肝脏指数和脾脏指数(4.75%±0.80%,0.43%±0.07%)比较，第6、9、11周尾蚴感染后小鼠肝脏指数（6.27%±0.56%，8.40%±1.43%，7.48%±1.32%，P＜0.05，P＜0.01）和脾脏指数（1.24%±0.37%，2.47%±0.46%，2.62%±1.23%，P＜0.05，P＜0.01）明显升高，而肾脏指数则没有明显变化。
2）与正常组（ALT：30.90±1.44，AST：114.00±5.07）相比，谷丙转氨酶（ALT）和谷草转氨酶（AST）在第3周开始明显升高（ALT：91.00±1.36，AST:232.10±2.54，P＜0.01），第6周达到高峰（ALT：176.22±7.08，AST:319.67±9.96，P＜0.01），在第9周（ALT：103.60±4.34，AST：197.40±5.58，P＜0.01）和第11周（ALT：50.13±3.12，AST：203.88±4.14，P＜0.01）下降但仍明显高于正常水平。
3）α-SMA和desmin免疫荧光双重染色发现，分离的HSC纯度为95%，细胞活性为90%。
4）MTT比色法显示不同浓度的AEA对原代HSC增殖的抑制率分别为：5μM组为5.14%±1.61%、10μM组为10.34%±3.22%、20μM组为14.12%±3.98%、40μM组为18.64%±3.67%、60μM组为23.86%±3.06%、80μM组为62.81%±7.75%、100μM组为70.39%±7.11%、120μM组为72.31%±6.71%，AEA的IC50值为79.06±9.38μM。
5）Annexin V-FITC/PI双标的流式细胞术的结果显示，不同浓度的AEA诱导原代HSC的死亡率分别为：0μM组为9.5%±3.19%、5μM组为9.8%±2.28%、10μM组为11.18%±1.82%、20μM组为12.2%±2.39%、40μM组为15.1%±1.88%、60μM组为45.85%±4.73%、80μM组为63.04%±7.00%、100μM组为73.32%±4.62%、120μM组为89.47%±8.84%。
6）DAPI染色的结果显示AEA处理的原代HSC的细胞核肿胀破裂，染色质固缩成颗粒状，部分细胞的核内容物溢出。
结论在尾蚴感染后的第6周，小鼠的肝脏组织出现嗜酸性肉芽肿和胶原纤维及虫卵的沉积，肝脏组织和脾脏组织逐渐肿大，尾蚴贴敷腹部皮肤的方法可成功建立早期及晚期血吸虫肝损伤小鼠模型。AEA可呈浓度依赖性地抑制原代HSC的增殖并促进其坏死。
目的探讨原代HSC胞膜的脂筏、大麻素受体(CB)及氧化应激对AEA诱导的原代HSC增殖坏死及MAPK信号通路的影响，对细胞因子IL-6，MCP-1， TNF-α及IL-1β表达的影响。
方法1)Western-blotting检测AEA与MAPK主要成员P-Erk1/2,P-P38和P-JNK1/2的时间及浓度的关系。
2)分别用胆固醇耗竭剂MCD破坏脂筏的完整性，CB1受体拮抗剂SR141716及CB2受体拮抗剂AM631阻断AEA与其相应受体的结合，抗氧化剂NAC清除自由基，应用Annexin V-FITC/PI双标的流式细胞术观察上述处理对AEA诱导的原代肝星状细胞死亡影响。
3)应用MTT观察MCD，SR141716和AM639及NAC对AEA诱导的原代HSC死亡率的影响。
4)Western-blotting观察MCD，SR141716和AM639及NAC对AEA诱导的MAPK信号通路激活及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)表达的影响。
5)RT-PCR检测MCD，SR141716和AM639及NAC对AEA诱导的细胞因子IL-6，MCP-1，TNF-α及IL-1β表达的影响。
结果1)AEA可呈时间和浓度依赖性地促进P-Erk1/2,P-P38及P-JNK1/2的表达：在AEA浓度为20μM的时候开始促进P-Erk1/2,P-P38及P-JNK1/2的表达，随浓度升高表达逐渐增加（P均＜0.05）。在AEA分别孵育15min,30min,1h,3h,6h时P-Erk1/2和P-P38的表达较高，而后逐渐下降。在15min,30min,1h时，P-JNK1/2的表达较高，而后逐渐下降。
2)MCD和NAC可降低AEA诱导的原代HSC的死亡率（P均＜0.05），而SR141716和AM630则无明显作用。
3)MCD和NAC可降低AEA对原代HSC增殖的抑制（P＜0.05），而大麻素受体则无明显作用。
4)MCD、SR141716和NAC可抑制AEA诱导的P-Erk1/2、P-P38及P-JNK1/2的表达（P均＜0.05），AM630仅能抑制P-P38的表达（P＜0.05），而上述处理因素均对原代HSC内caspase-3的表达无明显影响。
5)AEA能诱导IL-6的表达增加，MCD、SR141716、AM630及NAC均可降低AEA诱导的IL-6的表达（P均＜0.05）。AEA可抑制MCP-1的表达（P＜0.05），MCD、SR141716及NAC对AEA诱导的MCP-1表达则无明显的影响，而AM630则能明显促进MCP-1的表达（P＜0.05）。AEA能降低TNF-α和IL-1β的表达（P均＜0.05），MCD、SR141716及NAC可进一步抑制AEA诱导的TNF-α和IL-1β的表达（P均＜0.05），而AM630则增强AEA诱导的TNF-α和IL-1β的表达（P均＜0.05）。结论内源性大麻素AEA可通过呈浓度和时间依赖性地激活MAPK信号通路促进原代HSC的坏死。脂筏及氧化应激是AEA诱导原代HSC坏死和抑制其增殖的必要条件，而与其胞膜的特异性大麻素受体无关。AEA可能是体内调节细胞因子表达的免疫分子。
目的观察不同剂量的内源性大麻素AEA对原代HSC内ROS的影响，观察AEA和MCD对NADPH氧化酶亚基gp91-phox和p47-phox表达的影响，并观察脂筏、大麻素受体及NADPH氧化酶在AEA诱导原代HSC内ROS产生过程中的作用，
方法1）利用荧光探针DCFH-DA可自由进入HSC胞膜，细胞内的酯酶水解生成DCFH。而DCFH不能通透细胞膜，从而使探针被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。在流式细胞仪上检测DCF的强度可间接反映原代HSC内ROS的水平。
2）Western-blotting检测MCD对AEA诱导的原代HSC内NADPH氧化酶亚基gp91-phox和p47-phox表达的影响，计算各组目的条带与β-actin灰度值的平均比值反应蛋白表达量。
3）观察大麻素受体拮抗剂SR141716或AM630，MCD及NADPH氧化酶抑制剂DPI和Apocynin，抗氧化剂NAC对AEA诱导的原代HSC内ROS产生的影响。
结果1）随着内源性大麻素AEA浓度的升高，原代HSC内ROS的水平逐渐升高。不同浓度的AEA诱导的ROS水平分别为：0μM组4.4±1.52，5μM组24.52±7.35，10μM组28.31±5.87，20μM组47.07±7.92，40μM组61.22±10.69，60μM组89.99±16.35，80μM组106.48±19.58，100μM组95.93±13.74，120μM组96.78±14.83。
2）AEA可促进原代HSC内NADPH氧化酶亚基gp91-phox和p47-phox的表达（P均＜0.05），加入MCD后其表达明显降低（P＜0.05）。①gp91-phox：各组与β-actin的平均比值分别为阴性对照组0.13±0.01，20μM AEA组0.29±0.03，60μM AEA组0.35±0.02，60μM AEA+MCD组0.20±0.01。②p47-phox：各组与β-actin的平均比值分别为阴性对照组0.06±0.01，20μM AEA组0.40±0.04，60μM AEA组0.66±0.06，60μMAEA+MCD组0.36±0.03。
3）SR141716和AM630并不能阻止AEA诱导的原代HSC内ROS的产生，而加入MCD和NAC后AEA诱导的原代HSC内ROS的水平明显降低（P均＜0.05），加入NADPH氧化酶抑制剂DPI和Apocunin后AEA诱导的原代HSC内ROS产生虽然有所降低，但差异没有统计学意义（P＞0.05）。各组的ROS水平分别为DMSO组6.52±2.78，20μMAEA组48.75±6.81，60μMAEA组105.87±15.79，60μMAEA+SR141716组107.85±10.57，60μMAEA+AM630组108.57±19.38，60μMAEA+MCD组40.35±5.82，60μMAEA+DPI+Apocynin组94.39±21.94，60μM AEA+MCD组35.96±7.59。
结论①AEA可呈浓度依赖性地促进原代HSC内ROS的产生。②内源性大麻素AEA促进NADPH氧化酶亚基gp91-phox和p47-phox的磷酸化依赖原代HSC胞膜脂筏的完整性。③AEA通过促进NADPH氧化酶亚基的磷酸化激活该酶，促进原代HSC内ROS的产生，而抑制NADPH氧化酶的活性后并不能阻止AEA诱导的ROS产生，表明NADPH氧化酶并不是AEA诱导原代HSC产生的ROS的主要来源。
目的观察AEA对血吸虫肝纤维化小鼠肝功能和病理学的影响。
方法1）制备血吸虫肝纤维化小鼠模型，将不同剂量的AEA（5mg/kg和10mg/kg）分别腹腔注射入小鼠体内，1次/天，共4周，计算各组每只小鼠的肝脏指数、脾脏指数。
2）全自动生化仪检测各组小鼠血清内转氨酶ALT和AST的含量。
3）HE染色观察各组肝脏组织的病理学变化。
4）Masson染色观察胶原纤维的沉积情况，并用Image-pro-plus软件计算胶原纤维的含量。结果1）血吸虫肝纤维化小鼠模型制备成功。与正常小鼠相比，模型组、5mg/kgAEA组和10mg/kgAEA组的肝脏指数、脾脏指数明显升高（P均＜0.05）。与模型组比较，5mg/kg AEA组和10mg/kg AEA组的肝脏指数和脾脏指数则无明显的变化。
2）与正常组比较，模型组、5mg/kg AEA组和10mg/kg AEA组的ALT和AST的含量明显升高（P均＜0.05）。与模型组比较，5mg/kgAEA组和10mg/kgAEA组的ALT和AST明显升高，差别具有统计学意义（P均＜0.05），而5mg/kg AEA组和10mg/kgAEA组在ALT和AST则无明显统计学意义。
3）HE染色结果显示与正常小鼠的肝脏组织相比，模型组、5mg/kg AEA组和10mg/kg AEA组均出现虫卵在血管的沉积，急慢性嗜酸性肉芽肿的形成，炎症细胞的浸润。与模型组比较，5mg/kgAEA组和10mg/kgAEA组的急慢性嗜酸性肉芽肿结节和炎症细胞浸润的面积增加。
4）Masson染色显示，在正常小鼠的肝脏组织中除组成血管的胶原纤维外，无胶原纤维的沉积，在模型组、5mg/kgAEA组和10mg/kgAEA组出现明显的围绕血管呈同心圆状的胶原沉积，胶原纤维的含量明显增加（P均＜0.05），而与模型组相比，5mg/kg AEA组和10mg/kg AEA组胶原纤维的含量无明显的统计学意义。
结论短期应用AEA可加剧血吸虫肝纤维化模型小鼠的肝功能，不能阻止肝纤维化的发展。
目的观察日本血吸虫可溶性抗原对原代HSC纤维化指标MMP-9、TIMP-1和α-SMA及P38/JNK MAPK信号通路和AKT信号通路的影响。
方法1）荧光定量RT-PCR检测RNA水平不同剂量的可溶性血吸虫抗原对MMP-9、TIMP-1和α-SMA表达的影响。
2）Western-blotting检测蛋白水平不同剂量的可溶性血吸虫抗原对MMP-9、TIMP-1和α-SMA表达及P38/JNK MAPK信号通路和AKT信号通路的影响。结果1）在RNA水平，与阴性对照相比，可溶性血吸虫抗原在50μg/ml和250μg/ml时可明显促进α-SMA和TIMP-1的表达（P均＜0.05），降低MMP-9的表达（P均＜0.05）。
2）在蛋白水平，与阴性对照相比，可溶性血吸虫抗原在50μg/ml和250μg/ml时可明显促进α-SMA和抑制MMP-9的表达（P均＜0.05），而对TIMP-1的表达没有明显的影响。
3）在蛋白水平，可溶性血吸虫抗原从5μg/ml至250μg/ml均可促进P-P38的表达，以50μg/ml和250μg/ml较为明显（P均＜0.05），在15μg/ml、50μg/ml和250μg/ml时促进P-JNK1/2的表达（P均＜0.05），而只有在250μg/ml时才促进P-AKT的表达（P＜0.05）。
结论日本血吸虫可溶性抗原在肝纤维化的早期和晚期通过P38/JNK MAPK信号通路促进其发生和发展，在晚期则可同时激活AKT信号通路促进其发展。
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.
引文
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