HMGB1在狼疮性肾炎肾小球系膜细胞增殖中的作用及机制
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摘要
目的：本研究拟以狼疮性肾炎(lupus nephrits,LN)模型MRL/faslpr小鼠和小鼠系膜细胞为研究对象，探讨高迁移率族蛋白（high mobility groupprotein B1,HMGB1）及其相关信号通路在狼疮性肾炎肾小球系膜细胞增殖中的作用及其可能作用机制，并应用电穿孔技术进行小鼠活体转染，观测敲低HMGB1及其相关信号通路因子的表达对小鼠肾脏损伤和肾功能改善情况，为狼疮性肾炎的病因研究和靶向治疗提供依据。
方法：
1检测HMGB1，PCNA和TLR2在MRL/faslpr小鼠肾组织的表达
28周龄雄性MRL/MPJ小鼠和MRL/faslpr小鼠（体重：45-55g）各8只，分别设为对照组（Control组）和狼疮性肾炎组（LN组）。留取24h尿液及内眦动脉取血后处死小鼠，取肾脏皮质，部分置于4%多聚甲醛固定，用于光镜检测；部分置于液氮保存，用于冰冻切片和肾小球目的蛋白及RNA水平检测。常规病理染色技术观察肾小球形态结构；免疫荧光技术和Western blot检测肾皮质HMGB1、PCNA和TLR2蛋白的表达；显微切割技术提取肾小球组织，Real-time PCR检测肾小球HMGB1、PCNA和TLR2mRNA的表达；雷诺RT-9600半自动生化分析仪检测Scr、BUN和24h蛋白尿生化指标。
2敲低MRL/faslpr小鼠肾皮质HMGB1的表达对小鼠肾脏增殖水平及肾功能的影响
⑴以小鼠系膜细胞为研究对象，脂质体介导向细胞内转染shHMGB1质粒，Western blot和Real-time PCR检测shHMGB1质粒对小鼠系膜细胞HMGB1蛋白及mRNA表达水平的抑制作用。⑵28周龄雄性MRL/MPJ小鼠（体重：45-55g）6只设为对照组（Control组），18只等周龄等体重MRL/faslpr雄性小鼠随机分为狼疮性肾炎组（LN组），shNC干预组（LN+shNC组）和shHMGB1干预组（LN+shHMGB1组）。电穿孔技术向治疗组小鼠肾脏皮质转染干扰质粒。2周后留取24h尿液和内眦动脉取血后处死小鼠取肾脏皮质，部分置于4%多聚甲醛固定，用于常规形态学检测；部分皮质置于液氮保存，用于冰冻切片和肾皮质蛋白检测。常规病理染色技术检测小鼠组织形态结构；免疫荧光检测肾小球内HMGB1和PCNA蛋白的表达；Western blot和Real-time PCR检测肾皮质中HMGB1和PCNA蛋白及mRNA表达水平；雷诺RT-9600半自动生化分析仪检测Scr、BUN和24h蛋白尿生化指标。
3检测HMGB1对体外培养小鼠系膜细胞增殖水平的影响及其作用机制
小鼠系膜细胞在37℃、5%CO2条件下，以含10%胎牛血清的DMEM/F12(3:1)培养基培养。⑴为了探讨HMGB1对细胞增殖的影响，将细胞分别以50μg.L-1、100μg.L-1和200μg.L-1HMGB1刺激2、4、8和12h，并于收集细胞前1h加入10μmol.L-1BrdU，以ELISA技术检测小鼠系膜细胞的增殖水平。⑵根据⑴的实验结果，将MMC细胞随机分为对照组和100μg.L-1HMGB1刺激组，分别于刺激10min、20min、30min、40min、50min和1h时收集细胞，Western blot检测p85、p110、p-Aktser473、Akt、p-IκBa和IκBa蛋白的表达水平，免疫细胞化学检测NF-κB p65核转位情况。⑶细胞随机分为Control组、HMGB1刺激组、LPS+HMGB1组和TLR2AB（block antibody）+LPS+HMGB1组，先后加入5μg.mL-1TLR2AB(blockantibody)孵育1h，5μg.mL-1LPS孵育2h，100μg.L-1HMGB1或DMSO，继续孵育8h，并于收集细胞前1h加入10μmol.L-1BrdU，Western blot和Real-time PCR检测PCNA蛋白和mRNA表达情况，免疫细胞化学检测BrdU表达情况。⑷细胞随机分为Control组和HMGB1刺激组，100μg.L-1HMGB1孵育10min，免疫共沉淀结合Western blot检测TLR2蛋白和p85蛋白结合水平。⑸细胞随机分为Control组，HMGB1刺激组，LY294002+HMGB1组和DMSO+HMGB1组，先后加入30μmol.L-1LY294002或DMSO孵育1h和100μg.L-1HMGB1孵育8h，并于收集细胞前1h加入10μmol.L-1BrdU，Western blot和免疫细胞化学检测细胞增殖水平；或先后加入或不加30μmol.L-1LY294002孵育1h和100μg.L-1HMGB1孵育50min，免疫细胞化学检测NF-κB p65核转位水平。⑹细胞随机分为Control组， HMGB1刺激组， PDTC+HMGB1组和DMSO+HMGB1组，先后加入或不加30μmol.L-1PDTC孵育1h和100μg.L-1HMGB1孵育8h，并于收集细胞前1h加入10μmol.L-1BrdU，Western blot和免疫细胞化学检测细胞增殖水平；或先后加入或不加30μmol.L-1PDTC孵育1h和100μg.L-1HMGB1孵育30min，Western blot检测p-Aktser473表达水平。⑺细胞随机分为Control组和HMGB1刺激组，100μg.L-1HMGB1孵育90min，染色质免疫共沉淀检测NF-κB p65蛋白和CylinD1启动子序列的结合水平。
4敲低MRL/faslpr小鼠肾组织TLR2或Akt的表达对小鼠肾脏增殖水平及蛋白尿水平的影响
⑴以小鼠系膜细胞为研究对象，脂质体介导向细胞内转染shTLR2或shAkt质粒，Western blot和Real-time PCR检测质粒对小鼠系膜细胞TLR2或Akt蛋白及mRNA表达水平的抑制作用。⑵28周龄雄性MRL/MPJ小鼠（体重：45-55g）6只设为对照组（Control组），18只等周龄等体重MRL/faslpr雄性小鼠随机分为狼疮性肾炎组（LN组），shNC干预组（LN+shNC组）组和shTLR2干预组（LN+shTLR2组）或shAkt干预组（LN+shAkt组）。电穿孔技术向治疗组小鼠肾脏皮质转染干扰质粒。2周后留取24h尿液和内眦动脉取血后处死小鼠取肾脏皮质，部分置于4%多聚甲醛固定，用于常规病理学染色技术检测；部分皮质置于液氮保存，用于冰冻切片和肾皮质蛋白检测。HE染色检测小鼠组织形态结构；免疫荧光检测肾小球内PCNA和TLR2或Akt蛋白的表达；Western blot和Real-time PCR检测肾小球中PCNA和TLR2或Akt蛋白及mRNA表达水平；雷诺RT-9600半自动生化分析仪检测Scr、BUN、尿白蛋白生化指标。
结果：
1HMGB1、PCNA和TLR2在MRL/faslpr小鼠肾小球的表达上调
⑴光镜观察：与Control组相比，LN组小鼠肾小球体积增大，细胞数目增多。⑵免疫荧光检测显示，在Control组小鼠肾小球中，HMGB1只表达于细胞核中，而在LN组小鼠肾组织中HMGB1则同时表达于肾小球细胞的胞核和核外； PCNA阳性信号在Control组和LN组小鼠肾小球中都主要定位于细胞核，但在后者的表达强于前者；TLR2在Control组小鼠肾小球中无明显表达，而在LN组肾小球中出现明显的阳性表达，且阳性信号呈系膜状呈现。⑶Western blot结果表明，与Control组相比，LN组小鼠肾皮质HMGB1、PCNA和TLR2蛋白的相对表达量显著升高。⑷Real-time PCR检测结果显示，与Control组相比，LN组小鼠肾小球HMGB1、PCNA和TLR2mRNA的表达水平均明显升高。⑷肾功能：与正常组相比，狼疮组小鼠24h蛋白尿水平升高，而Scr和BUN变化不明显。
2敲低HMGB1表达有效降低MRL/faslpr小鼠肾小球增殖水平及蛋白尿水平
⑴shHMGB1质粒能够有效下调小鼠系膜细胞中HMGB1蛋白及mRNA表达水平：Western blot结果显示，与Control组相比，shHMGB1组小鼠系膜细胞的HMGB1蛋白的表达水平明显降低； Real-time PCR结果显示，shHMGB1组小鼠系膜细胞的HMGB1mRNA水平较Control组均明显下调。⑵活体电穿孔技术可有效的将质粒转染至小鼠肾皮质：冰冻切片荧光显微镜下观察结果提示，电穿孔转染后1天、7天和14天目的质粒在小鼠肾皮质均有荧光表达，其中第7天时表达最强，第14天表达较低。⑶shHMGB1质粒有效下调狼疮模型小鼠HMGB1蛋白和mRNA的表达：免疫荧光检测结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球HMGB1蛋白的表达明显降低，且主要定位于细胞核；Western blot结果显示，与LN组小鼠相比，shHMGB1组小鼠肾皮质中HMGB1蛋白的表达水平明显降低；Real-time PCR结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球的HMGB1mRNA表达水平明显降低。⑷敲低肾组织中HMGB1表达能够降低狼疮模型小鼠肾小球细胞增殖水平：肉眼观察，各组小鼠肾脏体积、质量均无明显变化。光镜下观察，与LN组相比，LN+shHMGB1组小鼠肾小球细胞细胞数目有所减少，但仍高于Control组；免疫荧光检测结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球PCNA蛋白的表达明显降低；Western blot结果显示，与LN组小鼠相比，shHMGB1组小鼠肾皮质中PCNA蛋白的表达水平明显降低；Real-timePCR结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球的PCNAmRNA表达水平明显降低。⑸沉默肾组织中HMGB1表达能够降低狼疮性肾炎模型小鼠蛋白尿水平：生化检测结果显示，与LN组小鼠相比，shHMGB1组小鼠24h蛋白尿量出现明显的降低，血肌酐和血尿素水平无明显差别。
3HMGB1通过PI3K/Akt/NF-Κb/Cylin D1信号通路上调体外培养小鼠系膜细胞的增殖水平
⑴HMGB1能够上调小鼠系膜细胞增殖水平：BrdU掺入法ELISA技术检测结果显示，100μg.L-1HMGB1或200μg.L-1HMGB1刺激2h后，系膜细胞增殖水平均逐渐升高，并于8h达到高峰，12h逐渐降低；50μg.L-1HMGB1各时间点对系膜细胞的促增殖作用均低于100μg.L-1HMGB1组；而100μg.L-1HMGB1和200μg.L-1HMGB1两组之间HMGB1作用8h细胞增殖水平无明显差别。⑵HMGB1的促增殖作用是TLR2依赖性的： BrdU掺入法细胞免疫荧光检测结果显示，与Control组相比，HMGB1刺激组系膜细胞中BrdU阳性表达率明显增高；与HMGB1刺激组相比， LPS+HMGB1组BrdU阳性细胞比例显著增加，而TLR2中和抗体能够降低BrdU阳性细胞比例。⑶HMGB1上调TLR2与p85蛋白的结合：免疫共沉淀结合Western blot结果提示，Control组TLR2与p85无明显结合，HMGB1刺激5min后，检测到较明显与TLR2结合的p85蛋白条带。⑷HMGB1能够激活小鼠系膜细胞的PI3K/Akt/NF-κB信号通路：Western blot结果显示，HMGB1刺激30min时，p-Aktser473蛋白表达水平瞬时升高，同时，p-IκBa表达水平升高，而IκBa表达开始降低；细胞免疫荧光检测结果显示，正常细胞NF-κBp65主要定位于细胞浆，HMGB1刺激50min后，NF-κBp65主要在细胞核表达。⑸HMGB1所介导的核转位的NF-κBp65与Cyclin D1启动子区域结合增强：染色质免疫共沉淀结果提示，HMGB1刺激90min后，PCR检测到明显的与NF-κBp65结合的Cylin D1DNA。⑹LY294002和PDTC均能够抑制HMGB1对小鼠系膜细胞的增殖作用：BrdU掺入法细胞免疫荧光检测结果显示，与HMGB1组相比，LY294002+HMGB1组和PDTC+HMGB1组BrdU阳性细胞比例降低，Western blot和Real-time PCR结果显示，与HMGB1组相比，LY294002+HMGB1组和PDTC+HMGB1组PCNA和Cyclin D1蛋白和mRNA表达水平降低。⑺LY294002能够部分抑制NF-κBp65的核转位而PDTC对Akt活化水平无明显影响：免疫荧光结果显示，与HMGB1组相比，LY294002+HMGB1组NF-κBp65蛋白在系膜细胞细胞核内的表达降低，定位于胞浆和胞核。Western blot结果提示，PDTC+HMGB1组p-Aktser473表达水平与HMGB1组无明显区别。
4敲低TLR2表达有效降低MRL/faslpr小鼠肾小球增殖水平及蛋白尿水平，敲低Akt则对肾小球增殖水平及蛋白尿水平无明显影响
⑴shTLR2质粒可有效下调小鼠系膜细胞和肾组织中TLR2蛋白和mRNA水平：Western blot结果显示，与Control组相比，shTLR2组小鼠系膜细胞的TLR2蛋白的表达水平明显降低；Real-time PCR结果显示，shTLR2组小鼠系膜细胞的TLR2mRNA水平明显将低。⑵shTLR2质粒能够降低肾组织中TLR2蛋白和mRNA表达：免疫荧光检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾小球的TLR2的表达水平明显降低；Western blot结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾皮质TLR2蛋白表达明显降低；Realtime-PCR结果显示，与LN组小鼠相比，shTLR2组小鼠肾小球的TLR2mRNA表达水平均明显降低。⑶沉默肾组织中TLR2表达可降低肾小球细胞增殖：肉眼观察，各组小鼠肾脏体积、质量均无明显变化。但光镜下观察，与LN组相比，LN+shTLR2组小鼠肾小球细胞数目有所减少，但仍高于Control组；免疫荧光检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾小球的PCNA同样表达于细胞核，但表达水平明显降低；Western blot结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾皮质PCNA蛋白表达明显降低；Realtime-PCR结果显示，与LN组小鼠相比，shTLR2组小鼠肾小球的PCNAmRNA表达水平均明显降低。⑷沉默肾组织中TLR2表达能够有效降低小鼠蛋白尿水平：生化检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠的24h蛋白尿量出现降低，血肌酐和血尿素无明显差别。⑸shTLR2质粒对小鼠肾皮质p-Aktser473表达水平无明显影响：免疫荧光检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾小球p-Aktser473蛋白表达水平无明显变化。⑹shAkt质粒可有效下调小鼠系膜细胞中Akt蛋白和mRNA水平：Western blot结果显示，与Control组相比，shAkt组小鼠系膜细胞的Akt蛋白的表达水平明显降低；Real-time PCR结果显示，shAkt组小鼠系膜细胞的Ak tmRNA水平明显将低。⑺shAkt质粒能够降低肾组织中Akt蛋白和mRNA表达：免疫荧光检测结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾小球的Akt的表达水平明显降低；Western blot结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾皮质Akt蛋白表达明显降低；Realtime-PCR结果显示，与LN组小鼠相比，shAkt组小鼠肾小球的AktmRNA表达水平均明显降低。⑻shAkt对小鼠肾小球增殖水平无明显改善：肉眼观察，各组小鼠肾脏体积，质量均无明显变化。光镜下观察，LN组和LN+shAkt组小鼠肾小球形态结构无明显改善；免疫荧光检测结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾小球的PCNA蛋白表达水平无明显改变；Western blot结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾皮质PCNA蛋白表达水平无明显改变；Realtime-PCR结果显示，与LN组小鼠相比，shAkt组小鼠肾小球PCNAmRNA表达水平无明显改变。⑼shAkt质粒对小鼠蛋白尿水平无明显影响：生化检测结果显示，与LN组小鼠相比， LN+shAkt组小鼠的24h蛋白尿量、血肌酐和血尿素均未出现明显差别。
结论：
1与正常对照组MRL/MPJ小鼠相比，狼疮性肾炎模型MRL/faslpr小鼠肾组织中HMGB1、PCNA和TLR2的表达水平明显升高，而采用电穿孔转染技术沉默肾组织中HMGB1和TLR2表达能够降低肾小球细胞增殖水平和24h蛋白尿水平，提示HMGB1可能通过TLR2相关途径上调系膜细胞增殖水平从而参与狼疮性肾炎发生的。
2HMGB1可以上调体外小鼠系膜细胞的增殖水平，TLR2参与了促增殖过程，而PI3K/Akt/NF-κB/CylinD1信号通路被激活；TLR2抑制可有效的阻滞HMGB1的促增殖作用及PI3K/Akt/NF-κB/Cylin D1信号通路的活化；LY294002和PDTC均可阻滞HMGB1的促增殖作用，同时LY294002有效阻断HMGB1引起的p65的核转位，而PDTC对HMGB1引起的PI3K/Akt活化无明显影响；提示HMGB1在体外通过与其受体TLR2结合激活PI3K/Akt/NF-κB/Cylin D1信号通路，推动细胞周期进程，促进系膜细胞增生。
3电穿孔技术可有效介导目的质粒在狼疮性肾炎模型小鼠肾脏的活体转染并可维持质粒的较长时间表达。
Objective: MRL/faslpr mice and MMC (mice mesangial cell) line wereused in this study to investigate the role and possible mechanism of HMGB1on the proliferation of MMC in lupus nephritis, and to deternmine whetherproliferation level of glomeruli and renal function of mice were inproved byknockdowning HMGB1, TLR2and Akt expression in renal tissue ofMRL/faslpr mice by electroporation technology; which will provide amimportant basis for targeted therapy of lupus nephritis.
Methods:
1The expression of HMGB1, TLR2and PCNA in glomeruli ofMRL/faslpr mice
Eight28-week male MRL/MPJ mice and MRL/faslpr mice (weight45-55g) were respectively designed as control group and LN (lupus nephritis)group.24h urine and angular venous blood were obtained for detecting renalfunction and renal cortex was collected for relevant detections. HE stainingwas used to observe the morphological structure of glomeruli; the expressionof HMGB1, TLR2and PCNA was detected by immunofluorescence techniqueand Western blot; laser capture microdissection method was used to collectthe renal glomeruli and Real-time PCR was used to determine the HMGB1,TLR2and PCNA mRNA expression;24h Upro (urine protein), BUN (bloodurea nitrogen) and Scr (serum creatinine) was detected by Rayto RT-9600semiautomatic biochemistry analyzer.
2The effect of shHMGB1on glomeruli proliferation level and renalfunction of MRL/faslpr mice
⑴The silencing effect of shHMGB1vector on HMGB1protein andmRNA in mice mesangial cell was indentified by Western blot and Real-timePCR.⑵Six28-week male MRL/MPJ mice was designed as Control group, eighteen28-week male MRL/faslpr mice was randomly divided into LN group,LN+shNC group and LN+shHMGB1group. Electroporation technology wasused to transfect vector in vivo in treatment group. Mice were sacrificed twoweeks later after24h urine and angular venous blood were obtained, and renalcortex was collected for relevant detections. HE staining and PCNA detectionwere used to evaluate the proliferation of glomeruli in mice. Expression ofHMGB1and PCNA was detected by immunofluorescence technique andWestern blot; and expression of HMGB1and PCNA mRNA were observed byReal-time PCR.24h urine protein, blood urea nitrogen (BUN) and serumcreatinine (Scr) was detected by Rayto RT-9600semiautomatic biochemistryanalyzer.
3The effect and its mechanism of HMGB1on proliferation level of micemesangial cell line
Mice mesangial cell line (SV40MES130) were cultured in DMEM/F12(3:1) medium (Gibco BRL) supplemented with10%fetal bovine serum(Gibco BRL). The cells were synchronised by culturing in serum-free mediumfor24h.⑴To detect the effect of HMGB1on cell proliferation, the cells wererandomly divided into control group and the HMGB1groups (containing50,100, and200μg.L-1mouse recombinant HMGB1protein). Then the cells werecollected at2,4,8, and12h after stimulation or not. The cells were mixedwith BrdU (10μmol.L-1, SIGMA) for2h before collection.⑵The cells werecollected at10,20,30,40,50, and60min after stimulation with HMGB1(100μg.L-1) to detect the expression of PTEN, p-Akt (p-Ser473and p-Thy308), Akt,p-IκBα, IκBα by Western blot, the nuclear translocation of NF-κBp65wasobserved by immunofluorescence.⑶Cells were randomly divided into controlgroup, HMGB1group, LPS+HMGB1group and TLR2AB+LPS+HMGB1group to detect the role of TLR2in HMB1-induced proliferation of micemesangial cell. Cells were combined with5μg.mL-1TLR2AB for1h,5μg.mL-1LPS for2h,100μg.L-1HMGB1for8h and10μmol.L-1BrdU for1h.The proliferation level was measured by the expression of PCNA and CylinD1detected by Western blot and Real-time PCR and the corporation level of BrdU detected by immunofluorescence.⑷Cells were randomly divided intothree groups (control group, DMSO group and HMGB1group), and collectedafter stimulated by DMSO or HMGB1for10min, immunoprecipitationcombine with Western blot was performed to detect the expression level ofp85protein that incorporated with TLR2.⑸LY294002, specific inhibitors ofthe PI3K/Akr pathway, was used to determine the role of PI3K/Akt pathway inHMGB1-induced proliferation. Cells were randomly divided into four groups(control, HMGB1, HMGB1+LY294002, and HMGB1+DMSO) to explore theeffect of LY294002on the proliferation and nuclear translocation ofNF-κBp65and the expression of PCNA, Cyclin D1. The cells in theHMGB1+PDTC group were pre-treated with LY294002(30μmol.L-1, SIGMA)for1h. The cells were collected at50min as well as8h after stimulation withHMGB1(100μg.L-1), and the nuclear translocation of NF-κBp65andproliferation level were detected by immunofluorescence, Western blot andReal-time PCR respectively.⑹PDTC, specific inhibitors of the NF-κBpathway, was used to detect the role of NF-κB pathway in HMGB1-dependentproliferation. The cells were randomly divided into four groups (control,HMGB1, HMGB1+PDTC, and HMGB1+DMSO) to explore the effect ofPDTC on the proliferation and nuclear translocation of NF-κBp65and theexpression of PCNA, Cyclin D. The cells in the HMGB1+PDTC group werepre-treated with PDTC (5μmol.L-1, SIGMA) for30min. The cells werecollected at20and50min as well as8h after stimulation with HMGB1(100μg.L-1), and the phosphorylation level of Akt (p-ser473), the nucleartranslocation of NF-κB p65and proliferation level were detected by Westernblot, Real-time PCR and immunofluorescence respectively.⑺Cells wererandomly divided into three groups (control group, DMSO group and HMGB1group) and collected after stimulated by DMSO or HMGB1for90min, andchromatin immunoprecipitation was performed to detect the combine level ofp65protein and NF-κBp65promoter.
4Effect of knockdowning TLR2and Akt expression on glomeruliproliferation level and renal function of MRL/faslpr mice
⑴The silencing effect of shTLR2and shAkt vector on TLR2or Aktprotein and mRNA in mice mesangial cell was indentified by Western blot andReal-time PCR.⑵Six28-week male MRL/MPJ mice was designed as Controlgroup, eighteen28-week male MRL/faslpr mice was randomly divided intoLN group, LN+shNC group and LN+shTLR2group (LN+shAkt group).Electroporation technology was used to transfect vector in vivo in treatmentgroup. The expression of TLR2(Akt)，p-Aktser473and PCNA was detectedby immuno fluorescence technique and Western blot; Real-time PCR was usedto detect the expression of TLR2, Akt and PCNA mRNA.
Results:
1The expression of HMGB1, TLR2and PCNA were up-regulated inrenal of MRL/faslpr mice.
⑴HE staining showed that the volume of glomeruli and the number ofglomeruli cells increased compared with control group.⑵Immunofluorescenceresult showed that HMGB1protein was mainly located in nuclear of glomerulicells in control group mice, however, extranuclear HMGB1expression wasdetected in the glomeruli of LN mice. PCNA protein was located in nuclear ofglomeruli cells in both control and LN group, but the expression of PCNA wassignificantly higher in LN group than that in control group. No obviouslyTLR2expression was detected in glomeruli of control group mice, but highTLR2fluorescence intensity was obtained in renal of LN group mice and thepositive signal was located in both nuclear and extranuclear of glomerulicells.⑶Western blot showed that the HMGB1, TLR2and PCNA proteinexpressions in renal cortex of LN group mice were up-regulated comparedwith control group.⑷The results of Real-time PCR showed that the HMGB1,TLR2and PCNA mRNA expression in glomeruli of LN group mice wereup-regulated compared with control group.
2shHMGB1effectively decreases the glomeruli proliferation level and24h urine protein of MRL/faslpr mice.
⑴The liposome-mediated gene transfer technology could successfullytransfer shNC and shHMGB1vector into mice mesangial cell line, and shHMGB1vector significantly decreased the expression of HMGB1proteinand mRNA of mice mesangial cells.⑵There was no obviously change inappearance, volume and quality of mice renal among different groups;however, HE staining showed tha the cell number of glomeruli decreased inshHMGB1group mice compared with LN group.⑶Immunofluorescence andWestern blot result showed that the expression of HMGB1and PCNA proteindecreased in glomeruli of mice of shHMGB1group compared with LN group,and Real-time PCR also showed the down-regulated HMGB1and PCNAmRNA in shHMGB1group.⑷The24h urine protein of mice in shHMGB1group decreased compared with LN group; and no significantly different wasobserved in BUN and Scr between two groups.
3HMGB1mediated the proliferation of mice mesangial cell byTLR2/PI3K/Akt/NF-κB/Cylin D1signal pathway.
⑴The BrdU assay showed that the proliferation level of MMC cells inthe100μg.L-1and200μg.L-1HMGB1-stimulated groups increased at8h and12h compared with control group, peaked at8h. But there was no significantdifference between100μg.L-1and200μg.L-1HMGB1-stimulated groups. Theimmunocytochemistry analysis revealed that the percentage of BrdU-positivecells in the HMGB1group was increased noticeably compared with thecontrol group at8h.⑵Western blot result showed that p-Aktser473(thephosphorylation of Akt ser473) was elevated at20min,, additionally, theexpression of IκBa was down-regulated and accompanied with up-regulatedexpression of p-IκBa at30min in HMGB1group. The positive expression ofNF-κB p65was located in the cytoplasm, and no positive staining wasobserved in the nuclei of the control MMC cells. However, the positiveexpression of the NF-κB p65subunit was located in the nuclei andsignificantly increased at50min exposed to HMGB1.⑶Immunofluorescenceresult showed that the positive BrdU ratio was significantly increased inLPS+HMGB1group compared with HMGB1group, but the BrdU posotiveexpression decreased pretreated with TLR2AB. In addition, the expression ofPCNA and Cylin D1protein and mRNA decreased in TLR2 AB+LPS+HMGB1group compared with LPS+HMGB1group.⑷Immunoprecipitation result showed that no western blot signal wasfound in control group and DMSO group, and an obviously fragment wasobtained from protein that immunoprecipitated with TLR2antibody in MMCstimulated by HMGB1.⑸The ChIP experiment showed that no PCR signalwas found in control group and DMSO group amplified by a pair primerspanning the kB2site in promoter of CyclinD1, but an obviously fragmentwas obtained from chromatin that immunoprecipitated with NF-κB p65antibody in MMC stimulated by HMGB1.⑹Immunofluorescence resultshowed thatthe positive BrdU ratio in LY294002+HMGB1group andPDTC+HMGB1group both decreased compared with HMGB1group; Westernblot and Real-time PCR result showed that the expression of PCNA and CylinD1protein and mRNA was down-regulated compared with HMGB1group.⑺Immunofluorescence result showed that the positive expression ofNF-κB p65protein located in both nuclear and cytoplasm of mice mesangialcell and nuclear location of NF-κBp65decreased compared with HMGB1group; However, PDTC did effect the level of pAkt-ser374induced byHMGB1by western blot.
4shTLR2other than shAkt effectively downregulated the glomeruliproliferation level and24h urine protein of MRL/faslpr mice
⑴The liposome-mediated gene transfer technology could successfullytransfer shNC, shTLR2and shAkt vector into mice mesangial cell line, andshTLR2and shAkt vector significantly decreased the expression of TLR2orAkt protein and mRNA of MMC cells.⑵There was no obviously change inappearance, volume and quality of mice renal among different groups,moreover, HE staining showed that the cell number in glomeruli decreased inshTLR2group compared with LN group, but no obviously change in renalcorex was detected between LN group and shAkt group.⑶Immunofluorescence, Western blot and Real-time PCR result showed that theexpression of TLR2or Akt protein and mRNA decreased in mice glomeruli ofshTLR2group or shAkt group compared with LN group. Importantly, the expression of PCNA protein and mRNA decreased in shTLR2groupcompared with LN group, but no significantly change was observed in shAktgroup.⑷The24h urine protein of mice in shTLR2group decreased comparedwith LN group; however, no significantly different was observed in BUN andScr between two groups. Interestingly, knockdown of Akt expression couldnot affect the renal function of MRL/faslpr mice.⑸Immunofluorescence resultshowed that there was no noticeable change in expression of p-Aktser473inglomeruli of shAkt group mice compared with LN group,.
Conclusions:
1The expression level of HMGB1, PCNA and TLR2increased inMRL/faslpr mice compared with control group, and knockdown of HMGB1and TLR2expression could decrease cell proliferation of glomeruli inMRL/faslpr and improve renal function, which indicates that HMGB1andTLR2-related signal pathway might play an important role in proliferation ofglomeruli of lupus nephritis.
2HMGB1up-regulated the proliferation level of MMC cell by aTLR2-dependent pathway, meanwhile PI3K/Akt/NF-κB/Cylin D1signalpathway was activated; in addition both LY294002and PDTC abolished theHMGB1-induced proliferation; LY294002block the nuclear translocation ofNF-κBp65, however, PDTC did not effect the activation of PI3K/Akt pathway.Above all, we concluded that HMGB1could mediate the proliferation ofMMC via TLR2-related pathway, and TLR2/PI3K/Akt/NF-κB/Cyclin D1signal pathway plays a crucial role in HMGB1-mediated development ofLN
3Electroporation technology could effectively mediate the transfection oftarget plasmid into renal of MRL/faslpr mice in vivo, in addition, the plasmidcould express in renal contex for at least two weeks.
方法：
1检测HMGB1，PCNA和TLR2在MRL/faslpr小鼠肾组织的表达
28周龄雄性MRL/MPJ小鼠和MRL/faslpr小鼠（体重：45-55g）各8只，分别设为对照组（Control组）和狼疮性肾炎组（LN组）。留取24h尿液及内眦动脉取血后处死小鼠，取肾脏皮质，部分置于4%多聚甲醛固定，用于光镜检测；部分置于液氮保存，用于冰冻切片和肾小球目的蛋白及RNA水平检测。常规病理染色技术观察肾小球形态结构；免疫荧光技术和Western blot检测肾皮质HMGB1、PCNA和TLR2蛋白的表达；显微切割技术提取肾小球组织，Real-time PCR检测肾小球HMGB1、PCNA和TLR2mRNA的表达；雷诺RT-9600半自动生化分析仪检测Scr、BUN和24h蛋白尿生化指标。
2敲低MRL/faslpr小鼠肾皮质HMGB1的表达对小鼠肾脏增殖水平及肾功能的影响
⑴以小鼠系膜细胞为研究对象，脂质体介导向细胞内转染shHMGB1质粒，Western blot和Real-time PCR检测shHMGB1质粒对小鼠系膜细胞HMGB1蛋白及mRNA表达水平的抑制作用。⑵28周龄雄性MRL/MPJ小鼠（体重：45-55g）6只设为对照组（Control组），18只等周龄等体重MRL/faslpr雄性小鼠随机分为狼疮性肾炎组（LN组），shNC干预组（LN+shNC组）和shHMGB1干预组（LN+shHMGB1组）。电穿孔技术向治疗组小鼠肾脏皮质转染干扰质粒。2周后留取24h尿液和内眦动脉取血后处死小鼠取肾脏皮质，部分置于4%多聚甲醛固定，用于常规形态学检测；部分皮质置于液氮保存，用于冰冻切片和肾皮质蛋白检测。常规病理染色技术检测小鼠组织形态结构；免疫荧光检测肾小球内HMGB1和PCNA蛋白的表达；Western blot和Real-time PCR检测肾皮质中HMGB1和PCNA蛋白及mRNA表达水平；雷诺RT-9600半自动生化分析仪检测Scr、BUN和24h蛋白尿生化指标。
3检测HMGB1对体外培养小鼠系膜细胞增殖水平的影响及其作用机制
小鼠系膜细胞在37℃、5%CO2条件下，以含10%胎牛血清的DMEM/F12(3:1)培养基培养。⑴为了探讨HMGB1对细胞增殖的影响，将细胞分别以50μg.L-1、100μg.L-1和200μg.L-1HMGB1刺激2、4、8和12h，并于收集细胞前1h加入10μmol.L-1BrdU，以ELISA技术检测小鼠系膜细胞的增殖水平。⑵根据⑴的实验结果，将MMC细胞随机分为对照组和100μg.L-1HMGB1刺激组，分别于刺激10min、20min、30min、40min、50min和1h时收集细胞，Western blot检测p85、p110、p-Aktser473、Akt、p-IκBa和IκBa蛋白的表达水平，免疫细胞化学检测NF-κB p65核转位情况。⑶细胞随机分为Control组、HMGB1刺激组、LPS+HMGB1组和TLR2AB（block antibody）+LPS+HMGB1组，先后加入5μg.mL-1TLR2AB(blockantibody)孵育1h，5μg.mL-1LPS孵育2h，100μg.L-1HMGB1或DMSO，继续孵育8h，并于收集细胞前1h加入10μmol.L-1BrdU，Western blot和Real-time PCR检测PCNA蛋白和mRNA表达情况，免疫细胞化学检测BrdU表达情况。⑷细胞随机分为Control组和HMGB1刺激组，100μg.L-1HMGB1孵育10min，免疫共沉淀结合Western blot检测TLR2蛋白和p85蛋白结合水平。⑸细胞随机分为Control组，HMGB1刺激组，LY294002+HMGB1组和DMSO+HMGB1组，先后加入30μmol.L-1LY294002或DMSO孵育1h和100μg.L-1HMGB1孵育8h，并于收集细胞前1h加入10μmol.L-1BrdU，Western blot和免疫细胞化学检测细胞增殖水平；或先后加入或不加30μmol.L-1LY294002孵育1h和100μg.L-1HMGB1孵育50min，免疫细胞化学检测NF-κB p65核转位水平。⑹细胞随机分为Control组， HMGB1刺激组， PDTC+HMGB1组和DMSO+HMGB1组，先后加入或不加30μmol.L-1PDTC孵育1h和100μg.L-1HMGB1孵育8h，并于收集细胞前1h加入10μmol.L-1BrdU，Western blot和免疫细胞化学检测细胞增殖水平；或先后加入或不加30μmol.L-1PDTC孵育1h和100μg.L-1HMGB1孵育30min，Western blot检测p-Aktser473表达水平。⑺细胞随机分为Control组和HMGB1刺激组，100μg.L-1HMGB1孵育90min，染色质免疫共沉淀检测NF-κB p65蛋白和CylinD1启动子序列的结合水平。
4敲低MRL/faslpr小鼠肾组织TLR2或Akt的表达对小鼠肾脏增殖水平及蛋白尿水平的影响
⑴以小鼠系膜细胞为研究对象，脂质体介导向细胞内转染shTLR2或shAkt质粒，Western blot和Real-time PCR检测质粒对小鼠系膜细胞TLR2或Akt蛋白及mRNA表达水平的抑制作用。⑵28周龄雄性MRL/MPJ小鼠（体重：45-55g）6只设为对照组（Control组），18只等周龄等体重MRL/faslpr雄性小鼠随机分为狼疮性肾炎组（LN组），shNC干预组（LN+shNC组）组和shTLR2干预组（LN+shTLR2组）或shAkt干预组（LN+shAkt组）。电穿孔技术向治疗组小鼠肾脏皮质转染干扰质粒。2周后留取24h尿液和内眦动脉取血后处死小鼠取肾脏皮质，部分置于4%多聚甲醛固定，用于常规病理学染色技术检测；部分皮质置于液氮保存，用于冰冻切片和肾皮质蛋白检测。HE染色检测小鼠组织形态结构；免疫荧光检测肾小球内PCNA和TLR2或Akt蛋白的表达；Western blot和Real-time PCR检测肾小球中PCNA和TLR2或Akt蛋白及mRNA表达水平；雷诺RT-9600半自动生化分析仪检测Scr、BUN、尿白蛋白生化指标。
结果：
1HMGB1、PCNA和TLR2在MRL/faslpr小鼠肾小球的表达上调
⑴光镜观察：与Control组相比，LN组小鼠肾小球体积增大，细胞数目增多。⑵免疫荧光检测显示，在Control组小鼠肾小球中，HMGB1只表达于细胞核中，而在LN组小鼠肾组织中HMGB1则同时表达于肾小球细胞的胞核和核外； PCNA阳性信号在Control组和LN组小鼠肾小球中都主要定位于细胞核，但在后者的表达强于前者；TLR2在Control组小鼠肾小球中无明显表达，而在LN组肾小球中出现明显的阳性表达，且阳性信号呈系膜状呈现。⑶Western blot结果表明，与Control组相比，LN组小鼠肾皮质HMGB1、PCNA和TLR2蛋白的相对表达量显著升高。⑷Real-time PCR检测结果显示，与Control组相比，LN组小鼠肾小球HMGB1、PCNA和TLR2mRNA的表达水平均明显升高。⑷肾功能：与正常组相比，狼疮组小鼠24h蛋白尿水平升高，而Scr和BUN变化不明显。
2敲低HMGB1表达有效降低MRL/faslpr小鼠肾小球增殖水平及蛋白尿水平
⑴shHMGB1质粒能够有效下调小鼠系膜细胞中HMGB1蛋白及mRNA表达水平：Western blot结果显示，与Control组相比，shHMGB1组小鼠系膜细胞的HMGB1蛋白的表达水平明显降低； Real-time PCR结果显示，shHMGB1组小鼠系膜细胞的HMGB1mRNA水平较Control组均明显下调。⑵活体电穿孔技术可有效的将质粒转染至小鼠肾皮质：冰冻切片荧光显微镜下观察结果提示，电穿孔转染后1天、7天和14天目的质粒在小鼠肾皮质均有荧光表达，其中第7天时表达最强，第14天表达较低。⑶shHMGB1质粒有效下调狼疮模型小鼠HMGB1蛋白和mRNA的表达：免疫荧光检测结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球HMGB1蛋白的表达明显降低，且主要定位于细胞核；Western blot结果显示，与LN组小鼠相比，shHMGB1组小鼠肾皮质中HMGB1蛋白的表达水平明显降低；Real-time PCR结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球的HMGB1mRNA表达水平明显降低。⑷敲低肾组织中HMGB1表达能够降低狼疮模型小鼠肾小球细胞增殖水平：肉眼观察，各组小鼠肾脏体积、质量均无明显变化。光镜下观察，与LN组相比，LN+shHMGB1组小鼠肾小球细胞细胞数目有所减少，但仍高于Control组；免疫荧光检测结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球PCNA蛋白的表达明显降低；Western blot结果显示，与LN组小鼠相比，shHMGB1组小鼠肾皮质中PCNA蛋白的表达水平明显降低；Real-timePCR结果显示，与LN组小鼠相比，shHMGB1组小鼠肾小球的PCNAmRNA表达水平明显降低。⑸沉默肾组织中HMGB1表达能够降低狼疮性肾炎模型小鼠蛋白尿水平：生化检测结果显示，与LN组小鼠相比，shHMGB1组小鼠24h蛋白尿量出现明显的降低，血肌酐和血尿素水平无明显差别。
3HMGB1通过PI3K/Akt/NF-Κb/Cylin D1信号通路上调体外培养小鼠系膜细胞的增殖水平
⑴HMGB1能够上调小鼠系膜细胞增殖水平：BrdU掺入法ELISA技术检测结果显示，100μg.L-1HMGB1或200μg.L-1HMGB1刺激2h后，系膜细胞增殖水平均逐渐升高，并于8h达到高峰，12h逐渐降低；50μg.L-1HMGB1各时间点对系膜细胞的促增殖作用均低于100μg.L-1HMGB1组；而100μg.L-1HMGB1和200μg.L-1HMGB1两组之间HMGB1作用8h细胞增殖水平无明显差别。⑵HMGB1的促增殖作用是TLR2依赖性的： BrdU掺入法细胞免疫荧光检测结果显示，与Control组相比，HMGB1刺激组系膜细胞中BrdU阳性表达率明显增高；与HMGB1刺激组相比， LPS+HMGB1组BrdU阳性细胞比例显著增加，而TLR2中和抗体能够降低BrdU阳性细胞比例。⑶HMGB1上调TLR2与p85蛋白的结合：免疫共沉淀结合Western blot结果提示，Control组TLR2与p85无明显结合，HMGB1刺激5min后，检测到较明显与TLR2结合的p85蛋白条带。⑷HMGB1能够激活小鼠系膜细胞的PI3K/Akt/NF-κB信号通路：Western blot结果显示，HMGB1刺激30min时，p-Aktser473蛋白表达水平瞬时升高，同时，p-IκBa表达水平升高，而IκBa表达开始降低；细胞免疫荧光检测结果显示，正常细胞NF-κBp65主要定位于细胞浆，HMGB1刺激50min后，NF-κBp65主要在细胞核表达。⑸HMGB1所介导的核转位的NF-κBp65与Cyclin D1启动子区域结合增强：染色质免疫共沉淀结果提示，HMGB1刺激90min后，PCR检测到明显的与NF-κBp65结合的Cylin D1DNA。⑹LY294002和PDTC均能够抑制HMGB1对小鼠系膜细胞的增殖作用：BrdU掺入法细胞免疫荧光检测结果显示，与HMGB1组相比，LY294002+HMGB1组和PDTC+HMGB1组BrdU阳性细胞比例降低，Western blot和Real-time PCR结果显示，与HMGB1组相比，LY294002+HMGB1组和PDTC+HMGB1组PCNA和Cyclin D1蛋白和mRNA表达水平降低。⑺LY294002能够部分抑制NF-κBp65的核转位而PDTC对Akt活化水平无明显影响：免疫荧光结果显示，与HMGB1组相比，LY294002+HMGB1组NF-κBp65蛋白在系膜细胞细胞核内的表达降低，定位于胞浆和胞核。Western blot结果提示，PDTC+HMGB1组p-Aktser473表达水平与HMGB1组无明显区别。
4敲低TLR2表达有效降低MRL/faslpr小鼠肾小球增殖水平及蛋白尿水平，敲低Akt则对肾小球增殖水平及蛋白尿水平无明显影响
⑴shTLR2质粒可有效下调小鼠系膜细胞和肾组织中TLR2蛋白和mRNA水平：Western blot结果显示，与Control组相比，shTLR2组小鼠系膜细胞的TLR2蛋白的表达水平明显降低；Real-time PCR结果显示，shTLR2组小鼠系膜细胞的TLR2mRNA水平明显将低。⑵shTLR2质粒能够降低肾组织中TLR2蛋白和mRNA表达：免疫荧光检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾小球的TLR2的表达水平明显降低；Western blot结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾皮质TLR2蛋白表达明显降低；Realtime-PCR结果显示，与LN组小鼠相比，shTLR2组小鼠肾小球的TLR2mRNA表达水平均明显降低。⑶沉默肾组织中TLR2表达可降低肾小球细胞增殖：肉眼观察，各组小鼠肾脏体积、质量均无明显变化。但光镜下观察，与LN组相比，LN+shTLR2组小鼠肾小球细胞数目有所减少，但仍高于Control组；免疫荧光检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾小球的PCNA同样表达于细胞核，但表达水平明显降低；Western blot结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾皮质PCNA蛋白表达明显降低；Realtime-PCR结果显示，与LN组小鼠相比，shTLR2组小鼠肾小球的PCNAmRNA表达水平均明显降低。⑷沉默肾组织中TLR2表达能够有效降低小鼠蛋白尿水平：生化检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠的24h蛋白尿量出现降低，血肌酐和血尿素无明显差别。⑸shTLR2质粒对小鼠肾皮质p-Aktser473表达水平无明显影响：免疫荧光检测结果显示，与LN组小鼠相比，LN+shTLR2组小鼠肾小球p-Aktser473蛋白表达水平无明显变化。⑹shAkt质粒可有效下调小鼠系膜细胞中Akt蛋白和mRNA水平：Western blot结果显示，与Control组相比，shAkt组小鼠系膜细胞的Akt蛋白的表达水平明显降低；Real-time PCR结果显示，shAkt组小鼠系膜细胞的Ak tmRNA水平明显将低。⑺shAkt质粒能够降低肾组织中Akt蛋白和mRNA表达：免疫荧光检测结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾小球的Akt的表达水平明显降低；Western blot结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾皮质Akt蛋白表达明显降低；Realtime-PCR结果显示，与LN组小鼠相比，shAkt组小鼠肾小球的AktmRNA表达水平均明显降低。⑻shAkt对小鼠肾小球增殖水平无明显改善：肉眼观察，各组小鼠肾脏体积，质量均无明显变化。光镜下观察，LN组和LN+shAkt组小鼠肾小球形态结构无明显改善；免疫荧光检测结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾小球的PCNA蛋白表达水平无明显改变；Western blot结果显示，与LN组小鼠相比，LN+shAkt组小鼠肾皮质PCNA蛋白表达水平无明显改变；Realtime-PCR结果显示，与LN组小鼠相比，shAkt组小鼠肾小球PCNAmRNA表达水平无明显改变。⑼shAkt质粒对小鼠蛋白尿水平无明显影响：生化检测结果显示，与LN组小鼠相比， LN+shAkt组小鼠的24h蛋白尿量、血肌酐和血尿素均未出现明显差别。
结论：
1与正常对照组MRL/MPJ小鼠相比，狼疮性肾炎模型MRL/faslpr小鼠肾组织中HMGB1、PCNA和TLR2的表达水平明显升高，而采用电穿孔转染技术沉默肾组织中HMGB1和TLR2表达能够降低肾小球细胞增殖水平和24h蛋白尿水平，提示HMGB1可能通过TLR2相关途径上调系膜细胞增殖水平从而参与狼疮性肾炎发生的。
2HMGB1可以上调体外小鼠系膜细胞的增殖水平，TLR2参与了促增殖过程，而PI3K/Akt/NF-κB/CylinD1信号通路被激活；TLR2抑制可有效的阻滞HMGB1的促增殖作用及PI3K/Akt/NF-κB/Cylin D1信号通路的活化；LY294002和PDTC均可阻滞HMGB1的促增殖作用，同时LY294002有效阻断HMGB1引起的p65的核转位，而PDTC对HMGB1引起的PI3K/Akt活化无明显影响；提示HMGB1在体外通过与其受体TLR2结合激活PI3K/Akt/NF-κB/Cylin D1信号通路，推动细胞周期进程，促进系膜细胞增生。
3电穿孔技术可有效介导目的质粒在狼疮性肾炎模型小鼠肾脏的活体转染并可维持质粒的较长时间表达。
Objective: MRL/faslpr mice and MMC (mice mesangial cell) line wereused in this study to investigate the role and possible mechanism of HMGB1on the proliferation of MMC in lupus nephritis, and to deternmine whetherproliferation level of glomeruli and renal function of mice were inproved byknockdowning HMGB1, TLR2and Akt expression in renal tissue ofMRL/faslpr mice by electroporation technology; which will provide amimportant basis for targeted therapy of lupus nephritis.
Methods:
1The expression of HMGB1, TLR2and PCNA in glomeruli ofMRL/faslpr mice
Eight28-week male MRL/MPJ mice and MRL/faslpr mice (weight45-55g) were respectively designed as control group and LN (lupus nephritis)group.24h urine and angular venous blood were obtained for detecting renalfunction and renal cortex was collected for relevant detections. HE stainingwas used to observe the morphological structure of glomeruli; the expressionof HMGB1, TLR2and PCNA was detected by immunofluorescence techniqueand Western blot; laser capture microdissection method was used to collectthe renal glomeruli and Real-time PCR was used to determine the HMGB1,TLR2and PCNA mRNA expression;24h Upro (urine protein), BUN (bloodurea nitrogen) and Scr (serum creatinine) was detected by Rayto RT-9600semiautomatic biochemistry analyzer.
2The effect of shHMGB1on glomeruli proliferation level and renalfunction of MRL/faslpr mice
⑴The silencing effect of shHMGB1vector on HMGB1protein andmRNA in mice mesangial cell was indentified by Western blot and Real-timePCR.⑵Six28-week male MRL/MPJ mice was designed as Control group, eighteen28-week male MRL/faslpr mice was randomly divided into LN group,LN+shNC group and LN+shHMGB1group. Electroporation technology wasused to transfect vector in vivo in treatment group. Mice were sacrificed twoweeks later after24h urine and angular venous blood were obtained, and renalcortex was collected for relevant detections. HE staining and PCNA detectionwere used to evaluate the proliferation of glomeruli in mice. Expression ofHMGB1and PCNA was detected by immunofluorescence technique andWestern blot; and expression of HMGB1and PCNA mRNA were observed byReal-time PCR.24h urine protein, blood urea nitrogen (BUN) and serumcreatinine (Scr) was detected by Rayto RT-9600semiautomatic biochemistryanalyzer.
3The effect and its mechanism of HMGB1on proliferation level of micemesangial cell line
Mice mesangial cell line (SV40MES130) were cultured in DMEM/F12(3:1) medium (Gibco BRL) supplemented with10%fetal bovine serum(Gibco BRL). The cells were synchronised by culturing in serum-free mediumfor24h.⑴To detect the effect of HMGB1on cell proliferation, the cells wererandomly divided into control group and the HMGB1groups (containing50,100, and200μg.L-1mouse recombinant HMGB1protein). Then the cells werecollected at2,4,8, and12h after stimulation or not. The cells were mixedwith BrdU (10μmol.L-1, SIGMA) for2h before collection.⑵The cells werecollected at10,20,30,40,50, and60min after stimulation with HMGB1(100μg.L-1) to detect the expression of PTEN, p-Akt (p-Ser473and p-Thy308), Akt,p-IκBα, IκBα by Western blot, the nuclear translocation of NF-κBp65wasobserved by immunofluorescence.⑶Cells were randomly divided into controlgroup, HMGB1group, LPS+HMGB1group and TLR2AB+LPS+HMGB1group to detect the role of TLR2in HMB1-induced proliferation of micemesangial cell. Cells were combined with5μg.mL-1TLR2AB for1h,5μg.mL-1LPS for2h,100μg.L-1HMGB1for8h and10μmol.L-1BrdU for1h.The proliferation level was measured by the expression of PCNA and CylinD1detected by Western blot and Real-time PCR and the corporation level of BrdU detected by immunofluorescence.⑷Cells were randomly divided intothree groups (control group, DMSO group and HMGB1group), and collectedafter stimulated by DMSO or HMGB1for10min, immunoprecipitationcombine with Western blot was performed to detect the expression level ofp85protein that incorporated with TLR2.⑸LY294002, specific inhibitors ofthe PI3K/Akr pathway, was used to determine the role of PI3K/Akt pathway inHMGB1-induced proliferation. Cells were randomly divided into four groups(control, HMGB1, HMGB1+LY294002, and HMGB1+DMSO) to explore theeffect of LY294002on the proliferation and nuclear translocation ofNF-κBp65and the expression of PCNA, Cyclin D1. The cells in theHMGB1+PDTC group were pre-treated with LY294002(30μmol.L-1, SIGMA)for1h. The cells were collected at50min as well as8h after stimulation withHMGB1(100μg.L-1), and the nuclear translocation of NF-κBp65andproliferation level were detected by immunofluorescence, Western blot andReal-time PCR respectively.⑹PDTC, specific inhibitors of the NF-κBpathway, was used to detect the role of NF-κB pathway in HMGB1-dependentproliferation. The cells were randomly divided into four groups (control,HMGB1, HMGB1+PDTC, and HMGB1+DMSO) to explore the effect ofPDTC on the proliferation and nuclear translocation of NF-κBp65and theexpression of PCNA, Cyclin D. The cells in the HMGB1+PDTC group werepre-treated with PDTC (5μmol.L-1, SIGMA) for30min. The cells werecollected at20and50min as well as8h after stimulation with HMGB1(100μg.L-1), and the phosphorylation level of Akt (p-ser473), the nucleartranslocation of NF-κB p65and proliferation level were detected by Westernblot, Real-time PCR and immunofluorescence respectively.⑺Cells wererandomly divided into three groups (control group, DMSO group and HMGB1group) and collected after stimulated by DMSO or HMGB1for90min, andchromatin immunoprecipitation was performed to detect the combine level ofp65protein and NF-κBp65promoter.
4Effect of knockdowning TLR2and Akt expression on glomeruliproliferation level and renal function of MRL/faslpr mice
⑴The silencing effect of shTLR2and shAkt vector on TLR2or Aktprotein and mRNA in mice mesangial cell was indentified by Western blot andReal-time PCR.⑵Six28-week male MRL/MPJ mice was designed as Controlgroup, eighteen28-week male MRL/faslpr mice was randomly divided intoLN group, LN+shNC group and LN+shTLR2group (LN+shAkt group).Electroporation technology was used to transfect vector in vivo in treatmentgroup. The expression of TLR2(Akt)，p-Aktser473and PCNA was detectedby immuno fluorescence technique and Western blot; Real-time PCR was usedto detect the expression of TLR2, Akt and PCNA mRNA.
Results:
1The expression of HMGB1, TLR2and PCNA were up-regulated inrenal of MRL/faslpr mice.
⑴HE staining showed that the volume of glomeruli and the number ofglomeruli cells increased compared with control group.⑵Immunofluorescenceresult showed that HMGB1protein was mainly located in nuclear of glomerulicells in control group mice, however, extranuclear HMGB1expression wasdetected in the glomeruli of LN mice. PCNA protein was located in nuclear ofglomeruli cells in both control and LN group, but the expression of PCNA wassignificantly higher in LN group than that in control group. No obviouslyTLR2expression was detected in glomeruli of control group mice, but highTLR2fluorescence intensity was obtained in renal of LN group mice and thepositive signal was located in both nuclear and extranuclear of glomerulicells.⑶Western blot showed that the HMGB1, TLR2and PCNA proteinexpressions in renal cortex of LN group mice were up-regulated comparedwith control group.⑷The results of Real-time PCR showed that the HMGB1,TLR2and PCNA mRNA expression in glomeruli of LN group mice wereup-regulated compared with control group.
2shHMGB1effectively decreases the glomeruli proliferation level and24h urine protein of MRL/faslpr mice.
⑴The liposome-mediated gene transfer technology could successfullytransfer shNC and shHMGB1vector into mice mesangial cell line, and shHMGB1vector significantly decreased the expression of HMGB1proteinand mRNA of mice mesangial cells.⑵There was no obviously change inappearance, volume and quality of mice renal among different groups;however, HE staining showed tha the cell number of glomeruli decreased inshHMGB1group mice compared with LN group.⑶Immunofluorescence andWestern blot result showed that the expression of HMGB1and PCNA proteindecreased in glomeruli of mice of shHMGB1group compared with LN group,and Real-time PCR also showed the down-regulated HMGB1and PCNAmRNA in shHMGB1group.⑷The24h urine protein of mice in shHMGB1group decreased compared with LN group; and no significantly different wasobserved in BUN and Scr between two groups.
3HMGB1mediated the proliferation of mice mesangial cell byTLR2/PI3K/Akt/NF-κB/Cylin D1signal pathway.
⑴The BrdU assay showed that the proliferation level of MMC cells inthe100μg.L-1and200μg.L-1HMGB1-stimulated groups increased at8h and12h compared with control group, peaked at8h. But there was no significantdifference between100μg.L-1and200μg.L-1HMGB1-stimulated groups. Theimmunocytochemistry analysis revealed that the percentage of BrdU-positivecells in the HMGB1group was increased noticeably compared with thecontrol group at8h.⑵Western blot result showed that p-Aktser473(thephosphorylation of Akt ser473) was elevated at20min,, additionally, theexpression of IκBa was down-regulated and accompanied with up-regulatedexpression of p-IκBa at30min in HMGB1group. The positive expression ofNF-κB p65was located in the cytoplasm, and no positive staining wasobserved in the nuclei of the control MMC cells. However, the positiveexpression of the NF-κB p65subunit was located in the nuclei andsignificantly increased at50min exposed to HMGB1.⑶Immunofluorescenceresult showed that the positive BrdU ratio was significantly increased inLPS+HMGB1group compared with HMGB1group, but the BrdU posotiveexpression decreased pretreated with TLR2AB. In addition, the expression ofPCNA and Cylin D1protein and mRNA decreased in TLR2 AB+LPS+HMGB1group compared with LPS+HMGB1group.⑷Immunoprecipitation result showed that no western blot signal wasfound in control group and DMSO group, and an obviously fragment wasobtained from protein that immunoprecipitated with TLR2antibody in MMCstimulated by HMGB1.⑸The ChIP experiment showed that no PCR signalwas found in control group and DMSO group amplified by a pair primerspanning the kB2site in promoter of CyclinD1, but an obviously fragmentwas obtained from chromatin that immunoprecipitated with NF-κB p65antibody in MMC stimulated by HMGB1.⑹Immunofluorescence resultshowed thatthe positive BrdU ratio in LY294002+HMGB1group andPDTC+HMGB1group both decreased compared with HMGB1group; Westernblot and Real-time PCR result showed that the expression of PCNA and CylinD1protein and mRNA was down-regulated compared with HMGB1group.⑺Immunofluorescence result showed that the positive expression ofNF-κB p65protein located in both nuclear and cytoplasm of mice mesangialcell and nuclear location of NF-κBp65decreased compared with HMGB1group; However, PDTC did effect the level of pAkt-ser374induced byHMGB1by western blot.
4shTLR2other than shAkt effectively downregulated the glomeruliproliferation level and24h urine protein of MRL/faslpr mice
⑴The liposome-mediated gene transfer technology could successfullytransfer shNC, shTLR2and shAkt vector into mice mesangial cell line, andshTLR2and shAkt vector significantly decreased the expression of TLR2orAkt protein and mRNA of MMC cells.⑵There was no obviously change inappearance, volume and quality of mice renal among different groups,moreover, HE staining showed that the cell number in glomeruli decreased inshTLR2group compared with LN group, but no obviously change in renalcorex was detected between LN group and shAkt group.⑶Immunofluorescence, Western blot and Real-time PCR result showed that theexpression of TLR2or Akt protein and mRNA decreased in mice glomeruli ofshTLR2group or shAkt group compared with LN group. Importantly, the expression of PCNA protein and mRNA decreased in shTLR2groupcompared with LN group, but no significantly change was observed in shAktgroup.⑷The24h urine protein of mice in shTLR2group decreased comparedwith LN group; however, no significantly different was observed in BUN andScr between two groups. Interestingly, knockdown of Akt expression couldnot affect the renal function of MRL/faslpr mice.⑸Immunofluorescence resultshowed that there was no noticeable change in expression of p-Aktser473inglomeruli of shAkt group mice compared with LN group,.
Conclusions:
1The expression level of HMGB1, PCNA and TLR2increased inMRL/faslpr mice compared with control group, and knockdown of HMGB1and TLR2expression could decrease cell proliferation of glomeruli inMRL/faslpr and improve renal function, which indicates that HMGB1andTLR2-related signal pathway might play an important role in proliferation ofglomeruli of lupus nephritis.
2HMGB1up-regulated the proliferation level of MMC cell by aTLR2-dependent pathway, meanwhile PI3K/Akt/NF-κB/Cylin D1signalpathway was activated; in addition both LY294002and PDTC abolished theHMGB1-induced proliferation; LY294002block the nuclear translocation ofNF-κBp65, however, PDTC did not effect the activation of PI3K/Akt pathway.Above all, we concluded that HMGB1could mediate the proliferation ofMMC via TLR2-related pathway, and TLR2/PI3K/Akt/NF-κB/Cyclin D1signal pathway plays a crucial role in HMGB1-mediated development ofLN
3Electroporation technology could effectively mediate the transfection oftarget plasmid into renal of MRL/faslpr mice in vivo, in addition, the plasmidcould express in renal contex for at least two weeks.
引文
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