Effect Of Total Saponins Of Panax Ginseng Combinated With The Rat Bone Marrow Mesenchymal Stem Cells On Insulin-producing Cells INS-1Impaired With High Glucose

Objective: Rat islet beta cell lines INS-1cells arecontinuously treated with high glucose until the cells’ viability andfunction is impaired. After that, we remove the high glucose, andindirectly co-culture the INS-1cells with rat bone marrow mesenchymalstem cells by trans-well system at different concentration of TSPG. Afterthe co-culture, we observe the expression of the key transcription factorsMafA and PDX-1mRNA which is related to INS-1cells’ function, theinsulin’s secretion under basic condition and after stimulated withglucose, and the content of TGF-β in extracellular fluid during co-culture,to find whether the using of rat bone marrow mesenchymal stem cellsalone or in combination with total saponins of panax ginseng could repairINS-1cells’ function which has been impaired by high glucose.Methods: firstly, rat islet beta cell lines INS-1and rat bone marrowmesenchymal stem cells (BM-MSCs) were cultured separately. Then,INS-1cells were treated continuously for72hours with high glucose.After that, the high glucose was removed, and the INS-1cells wereco-cultured with rat bone marrow mesenchymal stem cells by trans-wellsystem. Six groups were included in this experiment:1.Normal cellular group: normal INS-1cells without treatment with high glucose;2. INS-1cells impaired control group: INS-1cells treated with high glucose only;3. Co-culture groups without TSPG: INS-1cells treated with high glucose+BM-MSCs;4. Co-culture groups with50mg/L TSPG: INS-1cellstreated with high glucose+BM-MSCs+50mg/L TSPG;5. Co-culturegroups with100mg/L TSPG: INS-1cells treated with high glucose+BM-MSCs+100mg/L TSPG;6. co-culture groups with200mg/LTSPG:INS-1cells treated with high glucose+BM-MSCs+100mg/LTSPG. After96hours’ co-culture, total RNA and protein were got fromINS-1cells. Expression of PDX-1and MafA mRNA were detected byRT-PCR, expression of PDX-1protein was detected by Western Blot, andinsulin under basic condition and after stimulated with glucose weretested by ELISA, TGF-beta in extracellular fluid during co-culture istested by ELISA too. Result:(1) Establishment of INS-1cells’ impairedmodel by high glucose: after the treatment with glucose at theconcentration of20,30and40mmol/l, the viability of the cells in eachgroup decreased compared with the control group. Referring to otherrelevant experiment, the30mmol/L glucose which can impair the functionof INS-1cells obviously when the cells were treated for72hours isselected as the concentration of stimulation.(2) The toxic effects of TSPGon INS-1cells: after400and800mg/L TSPG intervened, the cells’ ODvalues decreased significantly (P <0.05) compared with normal group, suggesting that TSPG in this concentration range can inhibit INS-1cell’viability and proliferation. So we choose50,100and200mg/L TSPGconcentration as intervention concentration.(3) The expression of MafAmRNA and PDX-1mRNA in each group: compared with normal controlgroup, in the groups that had been treated with30mmol/l glucose theMafA mRNA and PDX-1mRNA’s expression were lower and thedifference was statistically significant. Compared with the impairedcontrol group, INS-1cells in the group treated with200mg/L TSPGexpress more MafA mRNA, while INS-1cells in the group treated withboth100and200mg/L TSPG express more PDX-1mRNA.(4) Theexpression of PDX-1protein in each group: the expression of thePDX-1on the protein were decreased in the groups that had been treatedwith30mmol/L glucose when compared to normal group. But co-culturegroup express more PDX-1protein than the impaired control group.Co-culture group without TSPG express less PDX-1protein than thegroup with200mg/L TSPG and the difference was statistically significant.(5) The TGF-beta content in extracellular fluid in each group: there is nosignificant difference between high glucose-impaired groups and normalcontrol group, but the co-culture group had more TGF-beta than theimpaired control group, and the differences were statistically significant.(6) INS-1cells’ basal insulin secretion and glucose-stimulated insulinsecretion: compared with normal control group, INS-1cells’ basal insulin and glucose-stimulated insulin were reduced in high glucose-treatedgroup. When compared to the impaired control group, INS-1cells inco-cultured group secret more insulin both in basal andglucose-stimulated condition. There is no significant difference between“with TSPG” and “without TSPG” in basal insulin secretion, but theinsulin in the groups with100and200mg/L TSPG increased significantlywhen stimulated by the glucose than that without TSPG. Conclusion:(1)BM-MSCs can promote the recovery of INS-1cells’ function which hasbeen impaired by high glucose, and this effect is at least in part caused byupregulating the expression of PDX-1protein.(2) A certain concentrationof TSPG combinated with BM-MSCs can further promote the recovery ofhigh-sugar-impaired INS-1cells’ function, and this effect is at least inpart caused by increasing expression of MafA mRNA, PDX-1mRNA andPDX-1protein.(3) The effect that BM-MSCs promotehigh-glucose-impaired INS-1cells’ function may be associated with therole of BM-MSCs’ paracrine secretion which secret TGF-β and othercytokines.