Figure 2
Attenuation of BMSC-induced antihyperalgesia after subtraction of Ccl4 and blocking CCR2. (a) CCL4 protein levels were reduced in P20 BMSCs. *p < 0.05, vs. P1 BMSCs, n = 4. (b) Primary BMSCs were transducted with Ccl4-shRNA plasmids containing GFP. Immunofluorescence staining of BMSC with GFP confirmed successful gene transfer. Scale = 10 μm. (c) Ccl4 mRNA expression was reduced in BMSCs transduced with Ccl4 shRNA lentivirus compared to Ctrl shRNA-treated cells (n = 3, RT-qPCR). *p < 0.05, vs. ctrl shRNA. (d) Attenuation of BMSC-induced antihyperalgesia by pretreatment of BMSCs with RNAi of Ccl4. Compared to Ctrl shRNA, Ccl4 shRNA pretreatment attenuated BMSC-produced antihyperalgesia in TL rats, as indicated by a reduction in EF50. Dashed lines connect data points not shown with the scale of the abscissa. Ipsi, ipsilateral to injury; Con, contralateral non-injured. (e) CCR2 protein levels were reduced in P20 BMSCs. *p < 0.05, vs. P1 BMSCs, n = 4. (f) Pretreatment of BMSC with RS 102895 (10 μM for 24 h), a CCR2b antagonist attenuated BMSC-produced antihyperalgesia. ##p < 0.01, ###p < 0.001; vs. Ctrl shRNA (C) and vs. Veh (D). (g) Pretreatment of BMSC with shRNA against Ccl4 (-Ccl4) or CCR2b antagonist RS 102895 (-CCR2) reduced BMSC-induced MOR upregulation in RVM in TL rats. *p < 0.05, vs. veh. Error bars in (a,c,e, and g) are S.E.M. and in (d and f) (n = 6–8) are 95% confidence intervals. Statistics: (a,c,e) Unpaired, Two-tailed Student’s t-test; (d,f) Two-way ANOVA followed by post-hoc comparisons with Bonferroni corrections; (g) One-way ANOVA followed by post-hoc comparisons with Bonferroni corrections. Cropped gel images are shown in 2a, e.g. See Supplementary Figure 9 for full-length blots.
