Role And Mechanism Of HMGB1on Cell Proliferation Of Glomeruli In Lupus Nephritis

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 miceEight28-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 mice⑴The 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 lineMice 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 mice⑴The 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 ofLN3Electroporation 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.