The Study Of Endogenous Interleukin-6Secretory Mechanism From Bone Marrow Mesenchymal Stem Cells

Part Ⅰ Toll-like receptor2signaling pathway affects theinterleukin-6secretion in bone marrow mesenchymal stemcellsObjective This study aimed to investigate the signaling pathway ofendogenous interleukin-6(IL-6) secretion in the bone marrowmesenchymal stem cells (MSC) using the specific agonist toll-like receptor2(TLR2), NFκB inhibitor and siRNA of targeting TLR2gene, and topreliminary elucidated the biologic function of IL-6from MSC followingthe MSC co-cultured with the PC12cells damaged by OGD.Methods Basis on our previous studies, the PC12cells were culturewith EBSS medium without glucose instead of its normal medium for4hours at37℃of a incubator with5%O2、95%N2and5%CO2, which builtthe OGD model of PC12cells. After injury, the oxygen andglucose-deprivation (OGD) damaged PC12cells were mix co-cultured withMSC for24hours. Meanwhile, the only OGD-treated PC12cells wereserved as the injured control group, and co-cultured with normal PC12cells as the co-culture control group. The expression level of apoptosis factorBax was detected by western blotting, and the concentrations of IL-6in themedium were tested by ELISA kit. The changes of TLR2, NFκB and IL-6mRNA and protein expression levels were used by real-time PCR andwestern blotting after treatments by TLR2agonist PGN-SA8ug/ml for24hours, siTLR2adenovirus for24hours or48hours, and the NFκB inhibitorPDTC for24hours, respectively.Results (1) The western blotting analysis showed that the level of Baxprotein expression in the OGD-treated PC12cells following co-culturedwith MSC was significantly lower than those in both the onlyOGD-damaged PC12cells and the co-cultured with normal PC12cellsgroups (P<0.05, P<0.01).(2) The IL-6release of the co-cultured with MSCgroup was sharply higher than in both the OGD-treated PC12cells and thegroup of co-cultured with normal PC12cells groups by ELISA.(3) Usingreal-time PCR technique, the levels of TLR2mRNA expression was readilyinduced by PGN-SA and reduced by Ad-siTLR2(P<0.01，P<0.001), whichwere both statistically differences compared with the unteatment group(P<0.05，P<0.01，P<0.001). Furthermore, with the changes of TLR2mRNA expression, the levels of NFκB and IL-6mRNA expressions werealso correspondingly increased or decreased in the MSC.(4) Besidesthose above, the results detected by western blotting displayed that theagonist PGN-SA could specifically up-regulate the level of TLR2protein expression in the MSC (P<0.01) and lead to increase the levels of NFκBand IL-6protein expressions, which were statistically differences comparedto the untreatment group after the analysis of quantitative comparison usingactin normalization (P<0.01). After infected by the Ad-siTLR2, theexpression level of TLR2protein in the MSC was significantly decreased(P<0.01，P<0.001), and the levels of NFκB and IL-6protein expressionswere obviously suppressed compared to the Ad-RFP control group. Inaddition, the specific inhibitor PDTC of NFκB not only down-regulated theNFκB protein expression level (P<0.01, compare to the control group), butalso decreased IL-6protein expression level (P<0.05, compare to theAd-RFP group), which was not significantly impaired to TLR2expressionlevel in the MSC (P>0.05).Conclusion (1) MSC co-culture can down-regulated the expressionlevel of apoptosis factor Bax in the OGD-treated PC12cells, suggestingthat MSC has the recovery function to the injured PC12cells which maybeclosely associate with the high level of endogenous IL-6secretion in theMSC.(2) The PGN-SA and Ad-siTLR2can specifically change theexpression levels of TLR2, and also made coherent changes on the NFκBand IL-6expression levels in the MSC.(3) The PDTC can effectivelyinhibit NFκB and IL-6expressions in MSC, but not affect on the level ofTLR2expression in the MSC, indicating that the IL-6release from MSC isregulated by the TLR2/NFκB signal pathway. PART Ⅱ Effects of interleukin-6on the toll-like receptor2pathway and nerve restoration functionObjective This study aimed to investigate the regulation ofendogenous IL-6secreted by MSC to the TLR2/NFκB signal pathway byscreening siIL-6-MSC the stable cell line and the recombinant Ad-IL-6adenovirus, and to explore the mechanism of MSC secrete IL-6and revealthe MSC recovery function to the injured PC12cells.Methods After infected by Ad-IL-6adenovirus for24or48hours, thelevels of TLR2, NFκB and IL-6mRNA and protein expression in the MSCwere detected by real-time PCR and western blotting. To obtain thesiIL-6-MSC cell line, rat specific siIL-6lentivirus were infected into theMSC, and then the cells were selected by5ug/ml puromycin. ThesiIL-6-MSC were co-cultured with the OGD injuryed PC12cells for24hours, and GFP-MSC coculture was served as co-culture control group.The expression levels of Bax were detected by western blotting, and thechanges of resting membrane potential in the PC12cells of the both groupswere tested by whole-cell patch clamp. Results (1) The expression levels of IL-6mRNA and protein in theMSC infected by Ad-IL-6adenovirus were significantly up-regulated(P0.01，P0.001), but no changes in both the TLR2and NFκB expression.(2) The stable cell line siIL-6-MSC screened by puromycin could observeabove85%strong green fluorescence under a flurescent microscopy.Further detection found that expression levels of IL-6mRNA and proteinwere significantly decreased（P0.05，P0.001），however, the levels ofTLR2and NFκB expressions were not statistical changes in thesiIL-6-MSC.(3) Following the OGD-treated PC12cells co-cultured withsiIL-6-MSC, the expression level of Bax was obviously increasedcompared to that in the GFP-MSC co-culture control group（P0.01）. Theresting membrane potential of OGD damaged PC12cells followingco-cultured with siIL-6-MSC was significantly higher than that of theGFP-MSC co-culture control group by whole-cell patch clamp, suggestingthat the MSC recovery function to the OGD-treated PC12cells werediminished when the expression level of IL-6was deceased in the MSC,Conclusion (1) The MSC cell line with stable expression of siIL-6issuccessfully screened.(2) The increase or decrease of IL-6expression levelin the MSC cannot lead to the changes the TLR2and NFκB expressions,further indicating that endogenous IL-6secretion in the MSC is regulatedby the TLR2/NFκB signal pathway, and IL-6release cannot be feedback tomodulate TLR2/NFκB pathway in the MSC.(3) With the decrease of IL-6 expression level, the improving restoration function of MSC to thedamaged PC12cells is diminished, demonstrating that the biologicalfunction of endogenous IL-6from MSC is anti-apoptosis and recovery theactivity of injured neuron.