[Role of HMGB1-RAGE/TLRs-NF-κB signaling pathway on bone mesenchymal stem cells transplantation therapy for lipopolysaccaride-induced coagulation disorder rats].

OBJECTIVE: To determine the effect of bone mesenchymal stem cells (BMSCs) in transplantation therapy for lipopolysaccharide (LPS)-induced coagulation disorder and the underlying mechanism of high mobility group protein B1-receptors for advanced glycation end products/Toll-like receptors-nuclear factor-κB (HMGB1-RAGE/TLRs-NF-κB) signaling pathway.

METHODS: BMSCs of female Sprague-Dawley (SD) rats ageing 4-5 weeks old were extracted and cultivated in vitro, and the fourth-passaged BMSCs phenotype was identified by flow cytometry for transplantation in the following experimental study. The rats were randomly divided into normal saline (NS) control group, LPS group, and BMSC group according to the random number table with 15 rats in each group. Coagulation disorders model was reproduced by injection of 1 mg/kg LPS via saphenous vein, and the rats in the NS control group was injected with equal volume NS. Those in the BMSC group were infused BMSC 0.5 mL containing 1×106 cells via tail vein at 2 hours after LPS injection, and the rats in other groups were injected with equal volume NS. Abdominal aorta blood was collected at 1, 3 and 7 days post operation. Coagulation indexes such as platelet count (PLT), platelet volume distribution width (PDW), mean platelet volume (MPV), plateletcrit (PCT), platelet large cell ratio (P-LCR), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), international normalized ratio (INR), and fibrinogen (FIB) were determined. The mRNA levels and contents of HMGB1, RAGE, TLR2/4 and NF-κB were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.

RESULTS: (1) The cells cultured in vitro were spindle shaped or flat. The fourth-passaged BMSCs phenotype was successfully identified by flow cytometry technology. (2) Coagulation indexes: compared with NS control group, PLT, PCT and FIB in LPS group were significantly decreased, PDW, MPV, P-LCP, and INR were significantly increased, and APTT, PT, and TT were significantly prolonged from the first day. Furthermore, those in LPS group were gradually ameliorated with prolongation of LPS induction time. The coagulation function abnormality induced by LPS was reversed by BMSCs with significant difference at 1 day as compared with LPS group [PLT (×109 /L): 398.8±17.9 vs. 239.1±15.8, PCT (%): 0.35±0.04 vs. 0.23±0.06, FIB (g/L): 1.7±0.6 vs. 0.8±0.1, PDW (%): 12.4±1.6 vs. 16.2±1.5, MPV (fl): 11.0±1.6 vs. 13.7±1.1, P-LCP (%): 13.0±2.1 vs. 15.3±2.7, INR: 1.52±0.17 vs. 1.82±0.19, APTT (s): 66.3±4.1 vs. 89.5±4.5, PT (s): 18.3±0.7 vs. 25.1±1.9, TT (s): 87.5±7.8 vs. 115.0±9.7, all P < 0.05], till 7 days. (3) HMGB1-RAGE/TLRs-NF-κB signaling pathway related molecules: compared with NS control group, the mRNA expressions and contents of HMGB1, RAGE, TLR2/4 and NF-κB were significantly increased in LPS group from the first day. However, the mRNA expressions and contents of the molecules in LPS group were gradually decreased with prolongation of LPS induction time. After BMSC intervention, the mRNA expressions and contents of molecules at 1 day were significantly lower than those of LPS group [HMGB1 mRNA (2-ΔΔCt ): 10.77±0.04 vs. 24.51±3.69, HMGB1 content (μg/L): 0.48±0.01 vs. 0.95±0.06; RAGE mRNA (2-ΔΔCt ): 11.57±1.11 vs. 18.08±0.29, RAGE content (μg/L): 0.73±0.04 vs. 1.37±0.06; TLR2 mRNA (2-ΔΔCt ): 2.60±0.22 vs. 12.61±0.27, TLR2 content (μg/L): 0.81±0.03 vs. 1.59±0.09; TLR4 mRNA (2-ΔΔCt ): 2.95±0.52 vs. 4.06±0.11, TLR4 content (μg/L): 0.80±0.09 vs. 1.18±0.11; NF-κB mRNA (2-ΔΔCt ): 1.29±0.06 vs. 7.79±0.25, NF-κB content (μg/L): 1.22±0.24 vs. 2.42±0.26, all P < 0.05], till 7 days.

CONCLUSIONS: BMSCs administration could ameliorate the coagulation function in LPS-induced coagulation disorder rats and these might be associated with HMGB1-RAGE/TLRs-NF-κB signaling pathway inhibition.