Pre-Treatment With Bone Marrow-Derived Mesenchymal Stem Cells Alleviated Organ Injury In Lps-Induced Dic Rat Model

Objective:We investigated the protective effect of bone marrow-derived mesenchymal stem cells (BMSCs) pre-treatment in lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) rat model.Methods:Part I BMSC Culture:We obtained primary BMSCs from allogeneic Wistar rats (Adult male, seven weeks, weight:160-170g.) and subcultured the BMSCs to obtain a higher purity. We observed the morphology of cultured BMSCs and investigated their proliferative capacity. To verify that the cultured cells were BMSCs, their cellular phenotypes were identified by flow cytometry. and we cultured them under specific conditions to observe whether they were able to differentiate to osteoblasts, adipocytes, or endothelial cells.Part II LPS-Induced Rat Models:A total of30Wistar rats (Adult male, seven weeks, weight:160-170g.) were divided into two groups:an experimental group (n=15) and a control group (n=15). All rats were anesthetized by an intraperitoneal injection of amobarbital sodium at a dose of30mg/kg. Then the rats in the experimental group received an injection of LPS at a dose of3mg/kg, slowly through the tail vein or femoral vein for1hour. The rats in the control group received an injection of normal saline, following the same injection procedure as in the experimental group. Four and eight hours later, blood samples were collected from the inferior vena cava and follow parameters were detected:Platelets (PLT), Fibrinogen (Fib), D-dimer (D-D), Activated partial thromboplastin time (APTT), Prothrombin time (PT). Eight hours after the LPS or saline injection, rats were sacrificed, and samples of their heart, liver, kidney, and lung were placed in10%formalin for24h. Organ tissue sections were processed by hematoxylin and eosin (HE) staining and Mallory phosphotungstic acid hematoxylin (PTAH) staining. The cellular morphology and fibrin microthrombosis were observed under the microscope for comparisons. In addition, we identified100glomerulus in every section, calculating the mean proportion of glomerulus containing fibrin microthrombosis. All these steps help verify the successful creation of an LPS-induced rat model.Part III Virus-Mediated Tracers:After the lentiviral vector (PGCL-GFP, pHelper1.0, pHelper2.0) was packaged, the virus supernatant was obtained and saved. We use5x104third-generation BMSCs, which grew in the virus fluid with a multiplicity of infection (MOI) of0.25,2.5,5,12.5, and25, under the following conditions: polybrene8g/ml,32℃,1000g, centrifugation2h; then cultured in12-well plates again with each well containing10%FBS DMEM1.5ml; medium was changed12h later and then changed every2-3days, when we observed the expression of fluorescence in BMSCs. Five days later, we counted the fluorescence in five visual fields in each hole to calculate the average transfection efficiency and quantify the relationship between MOI and transfected efficiency to calculate the optimum MOI value. Next, we injected1ml of culture medium containing approximately1x106of the day-5GFP-transfected BMSCs to10Wistar rats (Adult male, seven weeks, weight:160-170g) via tail vein or femoral vein for three times at intervals of24h. The rats were sacrificed at the end of24h after last injection, and heart tissues were saved in10%formalin for24h. Heart tissues were processed by HE and anti-GFP peroxidase staining to determine whether surviving BMSC were observed in heart tissues.Part IV Experimental Procedures:After the above experiments, we divided60Wistar rats (Adult male, seven weeks, weight:160-170g) equally into six groups; all rats received BMSCs or pure culture medium injections in tail or femoral veins for three times at intervals of24h, then LPS or saline were given at the end of24h after last injection (Shown as Table1). We collected blood samples from the inferioer vena cava prior to the injection and4h,8h after the LPS or saline infusion respectively, parameters were monitored as follows:PLT, Fib, D-D, APTT, PT, Tumor Necrosis Factor-a (TNF-α), Interferon-γ (IFN-γ), Interleukin-1(3(IL-1β), Creatinine (Cr), Alanine aminotransferase (ALT), Creatinine kinase-MB (CK-MB), Endothelin(ET). 8h after the LPS or saline infusion the rats were sacrificed, and samples of their heart, liver, kidney, and lung were placed in10%formalin for24h. Organ tissue sections were processed by hematoxylin and eosin (HE) staining and Mallory phosphotungstic acid hematoxylin (PTAH) staining. The cellular morphology and fibrin microthrombosis were observed under the microscope for comparisons.Table1. Experimental treatments in which rats were injected with1ml culture medium (with or without BMSCs) and1ml saline (with or without LPS). Each experimental group had ten rats.To evaluate protective effects of BMSCs pretreatment on LPS-induced DIC rats, we selected60Wistar rats (Adult male, seven weeks, weight:160-170g) and randomly divided them into three groups:the LPS injection group (10mg/kg), the BMSCs pretreatment (1x103/time x3times, at intervals of24h) and LPS injection group (10mg/kg), the high-dose BMSCs pretreatment (1x105/time x3times, at intervals of24h) and LPS injection group (10mg/kg). Rats received the BMSCs injection via the tail vein or femoral vein and the LPS via intraperitoneal injection. We observed the survival times of rats in the three groups within24h after LPS injection and analyzed these survival data.Part V To further clarify the mechanism of the the BMSCs-pretreated organ protection, the proliferative capacity of peripheral blood lymphocytes (PBMC) from rats which underwent BMSCs pretreatment with different doses were measured. And we research the influence of allogeneic BMSCs on PBMC proliferationin under LPS stimulation in vitro. Finally, we study the influence of differ BMSCs-pretreated dose on PBMC proliferationin under LPS stimulation in vitro. The changes of inflammatory cytokines released by lymphocye in medium also measured.Results:1. BMSCs grew rapidly in the first five days, and the speed of cellular proliferation peaked on day six. After day6, the speed was maintained with slight fluctuations. Overall, BMSCs displayed vigorous growth. Under certain conditions, BMSCs cells differentiated into endothelial cells, osteoblasts, and adipocytes. The detection result of flow cytometry indicated cells with the positive phenotype of CD29, CD44, CD10, and CD106and negative phenotype of CD34and CD45, which is in accordance with the characterization of cell molecular phenotype of BMSCs.2. Compared to the control group, the LPS injection group displayed a significantly lower plasma PLT quantities and Fib levels (P<0.01), significantly higher D-2dimer levels (P<0.01), prolonged times of APTT (P<0.01) and PT (P<0.01). The tissue sections of kidneys, lungs, livers, and hearts in all groups were processed by HE staining and PTAH staining, which indicated formation of microvascular thrombosis and cell injury in LPS injection group compared to control group. All these confirmed successful LPS-induced DIC rat models.3. Forty-eight hours after lentivirus transfected293T cells, a large number of fluorescent cells were observed, which indicated high transfection efficiency. Five days after the virus transfected BMSC, we observed the fluorescence expression from BMSCs, and BMSCs grew well. The transfection efficiency differed under different MOI conditions, and MOI=12.5displayed the best transfection efficiency. GFP-labeled BMSCs were injected for consecutive three days, then the heart tissue displayed a positive stain by anti-GFP peroxidase staining, suggesting that the surviving BMSCs existed in myocardial tissues.4.①Compared to the group without BMSCs pretreatment, the BMSCs-pretreated groups displayed significantly higher plasma PLT quantities (P<0.01) and Fib levels (P0.05), significantly lower D-2dimer levels (P<0.01), reduced prolonged-times of APTT (P0.05) and PT (P0.01).②The BMSC-pretreated group also displayed lower plasma levels of TNF-a (P＜0.01), IFN-y (P＜0.01), IL-1β (P＜O.05), ALT(P0.01), Cr. CK-MB (P<0.05), and ET (P0.01). We found that more protective effects occurred with more BMSCs pretreatment, when BMSCs pretreated number between3x106and4x106we could get the best results.③The tissue sections of kidneys, lungs, livers, and hearts in all groups were processed by HE staining and PTAH staining, which indicated reduced formation of microvascular thrombosis and alleviated cell injury in BMSCs-pretreated groups compared to un-pretreated group. This effect was directly related to the number of BMSCs in the pretreatment.④In addition, the24-hour survival rate differed among the treatment groups. Survival in the un-pretreated group (group1) was34.2%, while51.3%of rats in the1x103BMSC pretreatment group (group2) and64.5%of rats in the1x105BMSCs pretreatment group (group3) survived (P=0.02). Survival in the group without pretreatment was significantly different from both the low pretreatment group (P=0.011) and the high pretreatment group (P=0.001); survival rates of pretreated groups were not significantly different from each other (P=0.35).5.〤ompared to the control group, the proliferative capacity of PBMC from rats underwent BMSCs pretreatment significantly weakened under LPS stimulation.②LPS can significantly stimulate the proliferation of PBMC, but if cocultured with BMSCs, this stimulated effect were significantly weakened (P<0.05). Under transwell culture, we get the same effect (P<0.05); we also found lymphocytes released more inflammatory factors when they under the stimulation of LPS, but if they cocultured with BMSCs, the release of inflammatory cytokine was significantly reduced. the same in Transwell culture.③Coculture with BMSCs significantly inhibited the LPS-stimulated proliferation of PBMC, more BMSCs cocultured, more inhibited effects. Coculture with BMSCs also significantly reduced the LPS-stimulated release of inflammatory factors from PBMC, more BMSCs cocultured, lower concentrations of inflammatory cytokines in medium.Conclusions:We obtained BMSCs from bone marrow of rats by using adherent methods and mechanical scraping for BMSCs culture and multiplication in vitro. This approach can yield BMSCs-like cells with high purity, excellent growth rates, and normal morphology. We verified that these cells were BMSCs using the detection of cellular phenotype by cytometry and by inducing these cells to differentiate to other various cells. Furthermore, LPS injection can successfully build a LPS-induced DIC rat model, and lentiviral vectors can successfully transfect BMSCs and make them labeled with GFP. Finally, BMSCs pretreatment can effectively reduce organ damage, inhibit intravascular coagulation, and improve the survival rate of rats in LPS-induced DIC model rat. These protective effects were BMSCs number-dependent; the mechanism may be related to the BMSCs’adjustment to the secretion of proinflammatory cytokines. BMSCs inhibit the LPS-stimulated proliferation of PBMC,reduced the LPS-stimulated release of inflammatory cytokine from PBMC, these effect don’t rely on the direct contact between cell to cell.To some extent,these effect are BMCSs-dose-dependent.