Effects Of TLR2on Restore Of Hypoxic-ischemic Brain Damage Following MSCs Transplantation And Its Mechanism

Part â…  Mesenchymal stromal cells protect from apoptosis ofpheochromocytoma cells challenged by hydrogen peroxide.Objective: Immunoregulation of mesenchymal stromal cells (MSCs)is more efficient at restoring biologic function of injured tissue thantransdifferentiation during transplantation. However, the exact mechanismsand characteristics of MSCs regarding immunomodulation are stillunknown, especially in the damaged niche after hypoxic-ischemic insult.Through H2O2-treated PC12cells and MSCs co-culture system to elucidateMSCs neuroprotection of H2O2-challenged PC12cells through reducingapoptosis and releasing cytokines. Further, we detected the expressionchanges of TLR2and related signaling pathway in this process.Methods: To mimic hypoxic-ischemic brain injury in vivo, differentconcentrations of H2O2(1.0mM,2.5mM,5.0mM) were used to treat PC12cells for1hour in vitro, while the untreated PC12cells as control group.MSCs were co-cultured directly with the injured PC12cells for24hours. And5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfo-phenyl) tetrazolium, inner salt (MTS), lactose dehydrogenase (LDH) andnitric oxide (NO) assays, intracellular Ca2+and resting membrane potential(RMP) were analyzed. Apoptotic-associated genes and cytokine releaseswere detected by reverse transcriptase-polymerase chain reaction (RT-PCR)andenzyme-linked immunosorbent assay (ELISA).Results:（1）PC12cells expressed the neuronal stem cell markernestin and neuronal markers NSE and MAP-2, and immunofluorescentlystained results also showed that PC12cells expressed MAP-2.（2）Thedamage was most light in1.0mM group compared with the normal controlgroup, and this was indifference. With the increase in H2O2concentration,the PC12cells changed into round and dark cells from the translucent flatirregular shape, with more suspension, condensation and fragments in theculture medium at1h after exposure to H2O2. Morphological observationindicated that MSCs co-culture promoted the repair of nerve injury of PC12cells.(3) The MTS assay results showed that After treatment with2.5mMand5.0mM H2O2, viability of the PC12cells decreased to about50%and75%of the control. There was no significant difference between normalPC12cells and1.0mM H2O2-challenged PC12cells. Only in the MSCsco-culture group was the viability of the PC12cells after exposure to H2O2compared with the PC12cells without H2O2treatment.(4) As the converseof the MTS assay, LDH release and NO concentration in the PC12cells were induced significantly by H2O2, compared with the PC12cells withoutH2O2treatment. Through continuous monitoring of the MSCs and H2O2-challenged PC12cells co-culture, we also found that the levels of LDHrelease in both the1.0mM and2.5mM co-culture groups were notsignificantly difference compared with the MSCs co-culture control groupwithout H2O2treatment.(5) The concentration of NO among the threeco-culture groups following H2O2treatment were significantly repressedcompared with the co-culture control group without H2O2treatment.(6)There was no statistical difference in the numbers of Annexin-V-PI stainedcells between the normal PC12cells and the PC12cells injured with the1.0mM H2O2treatment. The numbers of injured PC12cells that underwentapoptosis and/or necrosis following2.5mM and5.0mM H2O2treatmentwere significantly increased compared with normal PC12cells and thePC12cells damaged with1.0mM H2O2.Meanwhile, the numbers ofAnnexin-V-EGFP-positive cells in the early phase of apoptosis obviouslyincreased after co-culture with MSCs for24h, and Annexin-V-PI-positivecells in the late phase of apoptosis were significantly reduced comparedwith the PC12-alone groups with2.5mM and5.0mM H2O2treatments.(7)The Ca2+in the PC12-alone groups with1.0mM and2.5mM H2O2challenge had an increased influx from the extracellular space after NMDAstimulation, contrary to the sharp decrease in the PC12-alone cells withoutH2O2treatment. However, the MSCs co-culture signifi-cantly decreased the concentration of intracellular Ca2+in the PC12-alone groups with2.5mM and5.0mM H2O2treatment following NMDA irritation. There was nostatistical difference between the MSCs co-culture groups with1.0mMH2O2and without H2O2treatment.(8) The RMP of the MSCs co-culturegroup with2.5mM H2O2treatment was decreased more than the MSCsco-culture groups with1.0mM H2O2and without H2O2treatment. Thetreatments with1.0mM and2.5mM H2O2failed to alter the RMP ofPC12-alone cells.(9) We examined changes in genes, such as for Bcl-2andcaspase-3, that are associated with apoptosis, in both the PC12-alone andMSCs co-culture groups following exposure to H2O2. Although the mRNAexpression levels of Bcl-2and caspase-3genes had no visible differencesamong the PC12-alone groups with the three different concentrations ofH2O2treatment, the MSCs co-culture substantially increased levels of Bcl-2mRNA expression and promptly decreased levels of caspase-3mRNAexpression in both the2.5mM and5.0mM H2O2groups.(10) we detectedthe paracrine effects of MSCs through cytokine release using an ELISA.IL-10release was significantly increased in the PC12-alone groups by both2.5mM and5.0mM H2O2challenge, and statistically decreased in thePC12cells of the same H2O2concentration treatments after co-culture withMSCs. It was surprising that IL-6sharply increased more than10-fold inall the MSCs co-culture groups compared with PC12alone, even though nodifferences were found in the PC12-alone group or the MSCs co-culture group with the different H2O2treatments. we also examined theinflammatory cytokines TNF-α and IFN-β, as well as chemokine IL-8and NGF. Significant differences were not observed in IFN-β, IL-8andNGF cytokines among all the groups.(11) Western Blotting results showedthat the expressions of TLR2and NFкB P65were increased with theincrease in H2O2concentration in the PC12-Alone injury group, while weredecreased in MSCs co-culture group.Conclusions:(1) MSCs co-cultured with PC12cells reduced H2O2neurotoxic injury, and may delay the apoptosis of injured PC12cells.(2)These findings indicate that MSCs have neuroprotective effects onsuppressing cell apoptosis via regulation of the H2O2-impairedmicroenvironment, partly through IL-6and IL-10cytokine production.(3)MSCs may through TLR2â†'NFкB P65signaling pathway to regulate theinjured local microenvironment. Part â…¡TLR2regulates function of MSCs transplantation invivo on HIBD.Objective: To identify the repair effect of MSCs through TLR2signalpathway to treatment HIBD during transplantation in vivo, and then to clarify the immunoloregulation of TLR2signal pathway in the process ofHIBD.Methods: One hundred and eighty wistar rats were randomly dividedinto sham operation group (the sham group), HIBD model group(the HIBDgroup), Pam3CSK4plus HIBD group (the Pam3CSK4group) and MSCstransplantation to treatment of HIBD group (the MSCs group). Inaccordance with the method of Rice, we gave rats hypoxic-ischemic braindamage at age of7days. The rats were only dissected the neck skin thensutured in the sham group, the rats in the Pam3CSK4group were given30μg Pam3CSK through intraperitoneal injection16h before creatingHIBD model, and the rats in the MSCs group were supplied for1.5×106rMSCs by intraperitoneal injection24h after HIBD. Neurocognitivefunction of animal in different groups was tested with Morris water mazeexperiment. The expression of TLR2, NFκB P65and Bax in hippocampustissue of rats in different groups at different time points was determined byReal-time PCR and Western Blotting. Furthermore, we detectedconcentration of IL-10in the hippocampus tissues of rats in every groupusing ELISA kit.Results (1) In the morris water maze test, the average latency toplatform of the HIBD group was significantly longer than that in the shamgroup (#P <0.05vs. sham group), and the average latency of the MSCsgroup was longer than which of the sham group, meanwhile was shorter than which of the HIBD group (#P <0.05vs. sham group,&P <0.05vs.HIBD group). In the platform exploratory experiment at6d, the averageresidence time of rats in target quadrant was the longest in the sham groupand was the shortest in the HIBD group, at the same time, that was in themiddle of the MSCs group, the differences of which were statisticallysignificant (#P <0.05group HIBD vs. sham group).(2) Real-time PCR andWestern Blotting results showed that, when time going on (3d,7d and14d),the expression of TLR2in hippocampus tissue of rats in the HIBD groupwas gradually increased, while the result of comparison between the threegroups at the same time point displayed that the expression level of TLR2was increased in the HIBD group than in the sham group (#P <0.05vs.sham group). The expression level of TLR2was decreased in the MSCsgroup compared with the HIBD group (&P <0.05vs. HIBD group).(3) Atthe same time, cytokine release of IL-10in left hippocampus tissue of ratsin the HIBD group was higher than that in the sham group. MSCstransplantation decreased the secretion of IL-10in left hippocampus tissue(#P <0.05in the HIBD group and MSCs group vs. sham group,&P＜0.05representation of MSCs group vs. HIBD group).(4) Compared with HIBDrats, TLR2specific agonist Pam3CSK4significantly increased the averagelatency to platform of rats in the Pam3CSK4group, and reduced theaverage residence time in target quadrant, also enhanced the expressionlevel of TLR2, NF κ B and Bax in left hippocampus tissue, while the secretion of IL-10in left hippocampus tissue was increased.Conclusions: MSCs transplantation to treatment HIBD can promoterestitution of the learning and memory function of rats. In the process ofHIBD and MSCs transplantation to treatment HIBD, TLR2plays animportant role in immune regulation through regulating the expression ofdownstream signaling molecule NFкB to influence the expression of Baxand cytokine release of IL-10. Part III The molecular mechanism of TLR2pathway on restoringneurologied injury.Objective: To investigate about the molecular mechanism of TLR2signal pathway in the process of MSCs repaired injury of nerve using TLR2specific agonist Pam3CSK4and Ad-siTLR2on the model of PC12cellsfollowing OGD and MSCs co-culture system.Methods: First, three different groups, normal control group, PC12cells following OGD group and MSCs co-culture with PC12cells groupwere set in the experiment. Normal culture medium was replaced withEBSS in the OGD group and MSCs co-culture group, and then the cells were cultured in a cabin of5%O2plus95%N2at37℃for6hours; MSCsgroup was in normal culture medium after OGD, then was cocultured withMSCs in cell culture box of5%CO2at37℃for24hours. Real-time PCRand Western Blotting method were used to detect expressions of TLR2,NFкB P65and Bax in three groups; at the same time, ELISA was used todetect release of secreting IL-10. Then, recombinant adenovirus vectorcontaining specific small interfering RNA (siRNA) targeting rat TLR2genewas constructed and identified in PC12cells. Further, TLR2specificagonist Pam3CSK4and Ad-siTLR2were added in OGD group and MSCsgroup, Pam3CSK4was added6hours before OGD, and Ad-siTLR2was atleast24hours before OGD. Then, mechanisms of TLR2signal pathway onimmunoloregulation was detected in the process of MSCs repaired injuryof nerve.Results:(1) Model of oxygen glucose deprivation was establishedsuccessfully, morphological results suggested that PC12cells refraction andshrinkage after OGD, and apoptosis was increased.(2) The expression ofTLR2was increased in PC12cells of OGD group compared with controlgroup and was lower in MSCs group than in OGD group, there wassignificant difference between the two groups (P <0.05vs. OGD group).The expressions of NFкB P65gene and protein were on equal pace withthe expression of TLR2, but there were no significant difference betweenevery group. The expression of Bax was increased in OGD group than in control group, and apoptosis was increased; and the expression of Bax wasdecreased in MSCs group (P <0.05vs. OGD group). Cytokine release ofIL-10in the culture supernatant of OGD group was increased significantly,furthermore there was about3times increasing compared with controlgroup, while it declined in MSCs group. At same time, secretion of IL-10was rarely in the culture supernatants of MSCs (P <0.05vs. normal controlgroup).(3) Expressions of TLR2and NFкB were activation or inhibitionwhen using Pam3CSK4and Ad-siRNA, and the difference is statisticallysignificant (P <0.05vs. normal control group or Ad-RFP control group).(4)Expressions of TLR2, NFκB P65and Bax gene and protein were increasedwhen using Pam3CSK4in OGD group, and the secretion of IL-10wasincreased; while expressions of TLR2, NFκB P65and Bax gene and proteinweakened when using Ad-siTLR2in OGD group, and secretion of IL-10was reduced.(5) When using TLR2agonists Pam3CSK4or Ad-siTLR2,apoptosis was further slower in MSCs co-cultured group compared withOGD group.Conclusions:(1) MSCs have neuroprotective effects on OGDtreatment PC12cells.(2) TLR2played an important role in immuneregulation through regulating the expression of downstream signalingmolecule NFкB P65to reduce the expression of Bax and cytokine releaseof IL-10on the model of PC12cells following OGD and MSCs co-culturesystem.(3) Cytokine IL-10may as a role of inflammatory factor in the process of MSCs repair OGD injured PC12cells.