Ultrasound-guided HUC-MSCs transplantation alleviates neuropathic pain in CCI rats: a mechanistic study based on microglia/macrophage polarization and the NLRP3 inflammasome.

Intravenous application of human umbilical cord mesenchymal stem cells alleviate neuropathic pain by suppressing microglia activation in rats

To investigate the effects of dexmedetomidine on chronic constriction injury (CCI)-induced neuropathic pain and to further explore its mechanism. A CCI rat model was established and treatment with dexmedetomidine. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were monitored at different time points, and the effects of hematoxylin-eosin staining on the sciatic nerve morphology of rats were observed. Immunohistochemical and immunofluorescence analyses were used to detect the expression of high mobility group box-1 (HMGB1) protein and glial fibrillary acidic protein (GFAP), and protein fluorescence intensity of GFAP in spinal cord tissue, respectively. Moreover, the expression of HMGB1 and Toll-like receptor-4/nuclear factor kappa-B (TLR4/NF-κB) pathway-related proteins were detected by western blot assay. To verify whether dexmedetomidine alleviates CCI-induced neuropathic pain by inhibiting HMGB1-mediated astrocyte activation and the TLR4/NF-κB signaling pathway, the rats were further treated with an HMGB1 activator or antagonist. Dexmedetomidine was found to improve the pathological changes of the sciatic nerve and alleviate pain in the CCI rats. The expression of HMGB1, GFAP, TLR4, TRAF6, MyD88, and p-P65 were greatly downregulated in the spinal cord tissues of the CCI rats. In addition, a further study showed that an HMGB1 activator can reverse the inhibition of neuropathic pain behaviors of dexmedetomidine. Overexpression of HMGB1 downregulated the PWMT and PWTL and enhanced the astrocyte activity and the TLR4/NF-κB signaling pathway in CCI rats. These results indicated that dexmedetomidine can alleviate neuropathic pain in CCI rats by inhibiting HMGB1-mediated astrocyte activation and the TLR4/NF-κB signaling pathway.

To investigate whether mammalian target of rapamycin (mTOR) signaling pathway is involved in peripheral nerve injury-induced hyperalgesia through activation of spinal dorsal astrocytes in rats. Methods: A total of 30 male Sprague-Dawley (SD) rats were randomly divided into 6 groups (n=5): the 1 day group (D1 group), the 4 days group (D4 group), the 7 days group (D7 group), the 14 days group (D14 group), the normal group and the sham group. The sciatic nerve chronic constriction injury (CCI) model was established in the D1, D4, D7 and D14 group. The normal group received no treatment while the sham group was only exposed the sciatic nerve. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at the 1st, 4th, 7th, and 14th day after CCI in the different groups. Lumbar spinal cord were harvested on the 1st, 4th, 7th and 14th day in the D1, D4, D7, D14 group correspondingly, which were harvested on the 14th day in the normal group and the sham group. Distribution of mTOR in rat spinal cord was assessed by immunohistochemistry. The expressions of mTOR mRNA and protein in the spinal cord in different groups were determined by real-time PCR and Western blotting, respectively. Another 30 male intrathecal catheterized SD rats were randomly divided into 6 groups (n=5): a blank group, a CCI group, a CCI+early rapamycin (RAPA) group, a CCI+early dimethylsulfoxide (DMSO) group, a CCI+ later RAPA group, and a CCI+later DMSO group. The blank group didn't received any treatment; The CCI group was carried out the treatment of CCI model in the left hind limbs. 10 μL of 1% RAPA was given to the CCI+early RAPA group intrathecally at 4 hours after CCI for 3 days; the CCI+later RAPA group were treated with the same dose of RAPA on the 7th days after CCI for 3 days; the CCI+early DMSO group and the CCI+later DMSO group were injected with the same volume of 4% DMSO at the corresponding time as controls. The PWTL and PWMT were measured before and after intrathecal catheterization, and every other day after CCI. The lumbar spinal cords were selected and the expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn were examined by immunohistochemistry in the 14th day after CCI. Results: The immunohistochemistry positive particles of mTOR were widely distributed in the cytoplasm of the normal spinal neurons. Compared with the base line, the PWMT in the D14 group on the 1st, 4th, 7th and 14th day after CCI were significantly lower, and the PWTL on the 4th, 7th and 14th day after CCI were also significantly lower (P<0.05 or P<0.01). The expressions of mTOR mRNA and protein in the CCI groups (D1, D4, D7 and D14 group) were significantly increased than those in the normal group (P<0.05 or P<0.01). Compared with the CCI+early DMSO group, the PWMT and PWTL in the CCI+early RAPA group were obviously increased on 4th, 6th, 8th, 10th, 12th or 14th day after CCI (P<0.05 or P<0.01); compared with the CCI+later DMSO group, the PWMT and PWTL in the CCI+later RAPA group were also significantly increased at the 8th, 10th or 14th day after CCI (P<0.01 or P<0.05). The GFAP immunohistochemistry positive area and absorbance value in the dorsal horn of the lumbar spinal cord in the CCI rats were decreased in the CCI+early RAPA group compared with the CCI+early DMSO group (P<0.05 or P<0.01), and which were also decreased in the CCI+later RAPA group compared with the CCI+later DMSO group (P<0.05 or P<0.01). Conclusion: mTOR signaling pathway may be involved in hyperalgesia induced by peripheral nerve injury via spinal astrocyte activation in the dorsal horn of the spinal cord.

High-voltage long-duration pulsed radiofrequency attenuates neuropathic pain in CCI rats by inhibiting Cav2.2 in spinal dorsal horn and dorsal root ganglion

Neuropathic pain (NP) is a common clinical chronic pain with very complex mechanisms. This study explored the function of activated Wnt signaling pathway in NP. A rat model of chronic constriction injury (CCI) was established. Different doses of IWP-2, a Wnt signal inhibitor, were intrathecally injected to observe the behavior indicators at different time-points, including the pain induced by mechanical stimulation and thermal stimulation. The mRNA and protein levels of Wnt-3a, Frizzled 4 and β-catenin in lumbar (L) 4–6 dorsal root ganglion (DRG) of rats in each group, as well as synaptic plasticity-related molecules in DRG region of rats were detected by RT-PCR and western blotting, respectively. Compared with Sham group and Naive group, paw withdrawal thermal latency and paw withdrawal mechanical threshold were significantly decreased after CCI, while synaptic plasticity was increased (P<0.05). Besides, activation of Wnt/β-catenin signaling pathway was observed in rats with CCI. We found that intrathecal injection of IWP-2 effectively relieved the pain behavior and reduced the synaptic plasticity in rats with neuropathic pain after CCI, suggesting that the inactivated Wnt/β-catenin signaling pathway might be the major mechanism responsible for this effect. Our data demonstrated that intrathecal injection of IWP-2 ameliorated neuropathic pain in CCI rats by inhibiting the Wnt/β-catenin pathway.

Inflammatory response regulation is a mechanism through which human umbilical cord mesenchymal stem cells (HUCMSCs) improve myocardial ischemia reperfusion injury (IRI); however, the timing of HUCMSC delivery to achieve maximum effectiveness is controversial. To investigate the effects of HUCMSC delivery on the acute inflammatory stage of IRI, we transplanted HUCMSCs or HUCMSCs with cyclosporin A (CsA) through the coronary artery simultaneously during ischemia reperfusion in pigs. Ferumoxytol-labeled HUCMSCs (HUCMSC), HUCMSCs with cyclosporin A (HUCMSC+CsA), and PBS (control) groups were investigated to evaluate the homing of transplanted cells and changes in infarct features, cardiac activity, and inflammatory response at three time points post-transplantation. Animals were sacrificed 2 weeks later for histological analysis of the hearts. We detected Prussian blue-dyed granules distributed around T lymphocyte clusters in the infarct area in the HUCMSC group. Infarct size and collagen deposition in the infarct area were lower in the HUCMSC group than in the control and HUCMSC+CsA groups. Cardiac function was mildly impaired in both the control and HUCMSC groups, whereas added CsA had a more severe impact. The levels of proinflammatory markers were lower in the HUCMSC group than in the control group at 24-h follow-up, and the difference was more significant after adding CsA. There were more CD3+ T lymphocytes and Foxp3+ Tregs in the HUCMSC group infarct area than in the other two groups. Proliferation rate of T lymphocytes was higher in the HUCMSC group than in the other two groups. Indirect co-culture experiments in vitro showed that MSCs promoted the generation of CD4+CD25+ Foxp3+Tregs through a paracrine mechanism. These results indicate that immediate intracoronary delivery of HUCMSCs after ischemia reperfusion can reduce acute myocardial IRI and promote myocardial repair, mainly through T lymphocyte interactions to regulate the intense inflammatory response during the acute inflammatory stage.

The inhibition of nitric oxide-activated poly(ADP-ribose) synthetase attenuates transsynaptic alteration of spinal cord dorsal horn neurons and neuropathic pain in the rat

Mesenchymal stem cells (MSCs) derived exosomes (Exos) are one of the most promising candidate for the treatment of this condition. However, the underlying molecular mechanism remains uncertain. Here we investigated the therapeutic effect of exosomal miR-181c-5p (ExomiR-181c-5p) on a rat model of neuropathic pain induced by sciatic nerve chronic constriction injury (CCI). In this study NP model was established using the CCI method. NP levels were assessed using PWT and PWL. Microarray analysis and RT-PCR were used to determine the relative expression of miR-181c-5p. MSC-derived exosomes were extracted using the total exosome isolation reagent characterized by WB and NTA. MiR-181c-5p was loading into Exos using electroporation. The inflammation response in microglia cells and CCI rats were assessed by ELISA assay respectively. Our study demonstrates that miR-181c-5p expression was obviously decreased in a time-dependent manner in CCI rats. MiR-181c-5p was effectively electroporated and highly detected in MSC-derived Exos. ExomiR-181c-5p internalized by microglia cells and inhibit the secretion of inflammation factors. ExomiR-181c-5p intrathecal administration alleviated neuropathic pain and neuroinflammation response in CCI rats. Taken together, ExomiR-181c-5p alleviated CCI-induced NP by inhibiting neuropathic inflammation. ExomiR-181c-5p may be a valid alternative for the treatment of neuropathic pain and has vast potential for future development.

SNHG16 aggravates chronic constriction injury-induced neuropathic pain in rats via binding with miR-124-3p and miR-141-3p to upregulate JAG1

The protective impact and unique mechanisms of human umbilical cord mesenchymal stem cells (HUC-MSCs) transplantation following hypoxic-ischemic (HI)-induced brain white-matter injury (WMI) were explored. To establish a WMI model, Sprague-Dawley rats with three days after birth underwent unilateral carotid artery ligation, followed by hypoxic exposure (8% oxygen and 92% nitrogen). Subsequently, HUC-MSC transplantation was performed into the lateral ventricle. Molecular and behavioral experiments were conducted to assess how it would influence NLRP3 inflammasome activation, M1/M2 microglial polarization, and spatial cognitive abilities. HUC-MSCs promoted myelin regeneration and improved spatial cognitive function by blocking NLRP3 inflammasome activation. Furthermore, HUC-MSCs modified microglial polarization away from the M1 phenotype by downregulating the expression of CD86 and iNOS proteins and attenuating the release of proinflammatory cytokines such as TNF-α and IL-1β. They promoted anti-inflammatory cytokine production, such as TGF-β and IL-10, and the upregulation of CD206 and Arg-1 protein expression, thereby helping microglia transition to the M2 phenotype. HUC-MSCs inhibited NLRP3 inflammasome activation by antagonizing TLR4 receptors, induced microglial polarization towards the M2 phenotype in neonatal rats with WMI. HUC-MSCs seem to be a promising therapeutic option for treating WMI in premature infants.

Microglia regulate the inflammation of the central nervous system and play a crucial role in the pathogenesis of depression. Moreover, Jmjd3 is involved in microglia polarization. Mounting studies reported the beneficial effects of human umbilical cord mesenchymal stem cells (HUC-MSCs) on myocardial infarction (MI), Unfortunately, its effects on MI-induced depression and its underlying mechanisms remain unclear. We aimed to investigate the antidepressant effects of HUC-MSCs and their impacts on microglia polarization. In the current study, the MI model was established by ligating the left anterior descending coronary artery. Mice were injected with HUC-MSCs or PBS through the tail vein 1week after the surgery. The sucrose preference test (SPT), tail suspension test (TST), and forced swim test (FST) were performed to evaluate depression-like behavior. Cardiac function and myocardial fibrosis were evaluated at the end of the experiments. Immunofluorescence, Western blot, ELISA, and qRT-PCR were used to detect the levels of Jmjd3 and microglia-related markers and inflammatory factors. HUC-MSC treatment significantly improved cardiac function, reduced the area of myocardial fibrosis, and alleviated depression-like behaviors induced by MI. HUC-MSCs inhibited the expression of Jmjd3 and promoted the switch of microglia in the prefrontal cortex, hypothalamus, and hippocampus from M1 to M2, thereby decreased the level of pro-inflammatory factors. HUC-MSCs have cardioprotective and potential anti-depressive effects induced by MI related to the inflammation improved by regulating Jmjd3 and microglial polarization.

Objective To study effect of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells (HUCMSCs) for model rats with diabetic foot ulcer (DFU), and analyzed the possible action mechanism of HUCMSCs therapy DFU. Methods 50 specific pathogen free (SPF)-level male rats were fed with high-fat beverage for 4 weeks. The DFU models were established by means of intraperitoneal injection of 1% streptozotocin (40 mg/kg) and steam fumigation. 30 successful model rats were divided into model group, high-dose HUCMSCs group (high-dose group: femoral artery injection with HUCMSCs 2.0×106/0.2 ml) and low-dose HUCMSCs group (low-dose group: femoral artery injection with HUCMSCs 1.0×106/0.2 ml), 10 rats/each group. Another 10 SPF-level male rats were selected and set as blank control group (blank group). Then wound healing rate, serum inflammatory factors, related cell growth factors, granulation tissue fibroblasts and newly born capillaries were compared between the groups. Results (1) Wound healing rate: The model group, low-dose group, high-dose group respectively were (26.31±4.32)%, (66.92±7.24)%, (74.74±8.32)%. low-dose group and high-dose group wound healing rate were significantly higher than model group (t=8.424, 11.201, P<0.01), high-dose group wound healing rate were significantly higher than low-dose group (t=3.148, P<0.05). (2) Serum inflammatory factors: model group, low-dose group and high-dose group interleukin (IL)-1, tumor necrosis factor-α (TNF-α), hs-CRP respectively were (6.12±0.25), (4.54±0.36), (4.20±0.33) ng/L; (29.45±3.12), (21.13±3.24), (18.20±2.65) ng/L; (0.61±0.08), (0.45±0.07), (0.37±0.06) ng/L. low-dose group and high-dose group IL-1, TNF-α, hs-CRP were significantly lower than model group (t=11.400, 5.849, 5.111, 14.666, 8.691, 8.222, P<0.05, P<0.01), high-dose group were significantly lower low-dose group (t=2.202, 2.214, 2.744, P<0.05); (3) Related cell growth factors: model group, low-dose group and high-dose group basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) respectively were (46.42±6.36), (61.35±7.20), (68.24±8.12) ng/L; (546.32±30.24), (722.36±32.17), (772.45±34.26) μg/L; (2.24±0.32), (3.65±0.41), (4.26±0.55) μg/L. Low-dose group and high-dose group bFGF, EGF, VEGF were significantly higher than model group (t=4.915, 12.609, 8.573, 6.690, 15.648, 10.039, P<0.05, P<0.01). high-dose group bFGF, EGF and VEGF were significantly higher than low-dose group (t=2.031, 3.740, 2.812, P<0.05); (4) Granulation tissue fibroblasts and newly born capillaries: model group, low-dose group and high-dose group granulation tissue fibroblasts and newly born capillaries were respectively (155.24±8.45), (278.65±9.12), (305.12±10.25) cells/visual field; (4.36±0.45), (6.45±0.62), (7.24±0.60)×10/μm2. The low-dose group and high-dose group were significantly higher than model group (t=31.389, 8.627, 35.679, 12.143, P<0.01), high-dose group were significantly higher than low-dose group (t=6.101, 2.896, P<0.05). Conclusion HUMSCs help to promote the ulcerative healing of DFU rats. Key words: Diabetic foot ulcer; Human umbilical cord mesenchymal stem cells; Inflammatory factor; Growth factor

Human umbilical cord mesenchymal stem cells (HUMSCs) are candidates for tissue engineering and may potentially be used for transdifferentiation into pancreatic endocrine cells. The adenoviral vector is effective in transducing genes into stem cells that are refractory to gene delivery by non‑viral approaches. qPCR was used to detect the pancreatic endogenous gene expression of HUMSCs transfected by islet cell-specific transcription factors (TFs). In the present study, using adenoviruses, the mouse TFs, pancreatic and duodenal homeobox 1 (pdx1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (mafa) and class B basic helix‑loop‑helix factor neurogenin 3 (ngn3), which are essential for pancreatic cell development, were introduced into HUMSCs to assess the expression of the pancreatic genes, glucagon, pdx1 and nk2 homeobox 2 (nkx2.2). When pdx1, mafa and ngn3 were cotransduced into HUMSCs, the expression of glucagon increased by 21‑fold at days 3 and 7 following transduction, while the endogenous pdx1 gene expression was increased by 15‑fold at day 3 and decreased by 70% at day 7. When mafa and ngn3 were cotransduced into HUMSCs, there was a 5‑fold increase in pdx1 gene expression at day 7, but no activation was observed at day 3. When mafa alone was introduced into HUMSCs, the pdx1 gene expression was elevated by 6‑fold at day 3 and decreased by 3‑fold at day 7. Transduction of ngn3 alone into HUMSCs induced nkx2.2 gene expression at day 3 but the expression levels were decreased at day 7. However, when pdx1 and ngn3 were cotransduced into HUMSCs, the expression levels of glucagon, pdx1 and nks2.2 were all lower than those observed with pdx1 or ngn3 transduction alone. These results suggested that the transduction of pdx1, mafa and ngn3 genes into HUMSCs induced the expression of the pancreatic genes, glucagon, pdx‑1 and nkx2.2, and that the expression was time dependent. In addition, different combinations of the TFs may demonstrate synergistic or antagonistic effects. This data may be beneficial for guiding future studies obtaining mature pancreatic endocrine cells from HUMSCs.

Human umbilical cord mesenchymal stem cells (HUMSCs) are highly proliferative and can be induced to differentiate into advanced derivatives of all three germ layers. Thus, HUMSCs are considered to be a promising source for cell-targeted therapies and tissue engineering. However there are reports on spontaneous transformation of mesenchymal stem cells (MSCs) derived from human bone marrows. The capacity for HUMSCs to undergo malignant transform spontaneously or via induction by chemical carcinogens is presently unknown. Therefore, we isolated HUMSCs from 10 donors and assessed their transformation potential either spontaneously or by treating them with 3-methycholanthrene (3-MCA), a DNA-damaging carcinogen. The malignant transformation of HUMSCs in vitro was evaluated by morphological changes, proliferation rates, ability to enter cell senescence, the telomerase activity, chromosomal abnormality, and the ability to form tumors in vivo. Our studies showed that HUMSCs from all 10 donors ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUMSCs from two of the 10 donors treated with 3-MCA displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. When these cells (tHUMSCs) were injected into immunodeficient mice, they gave rise to sarcoma-like or poorly differentiated tumors. Moreover, in contrast to HUMSCs, tHUMSCs showed a positive expression of human telomerase reverse transcriptase (hTERT) and did not exhibit a shortening of the relative telomere length during the long-term culture in vitro. Our studies demonstrate that HUMSCs are not susceptible to spontaneous malignant transformation. However, the malignant transformation could be induced by chemical carcinogen 3-MCA.

BackgroundPatients with systemic lupus erythematosus (SLE) suffer from a high incidence of premature ovarian failure, which might be due to cyclophosphamide gonadal toxicity, immune abnormalities, or other reasons. This study aimed to investigate whether the transplantation of human umbilical cord mesenchymal stem cells (HUC‐MSCs) can improve ovarian reserve function in lupus mice.MethodsWe used MRL/lpr mice to observe changes in ovarian structure and secretory function in SLE. Lupus mice and controls were injected with HUC‐MSCs at Weeks 12 and 16. We detected serum concentrations of the sex hormones estradiol (E2), follicle‐stimulating hormone (FSH), and anti‐Müllerian hormone (AMH), using enzyme‐linked immunosorbent assays. Hematoxylin and eosin staining showed ovarian tissue structure and enabled the counting of follicles. Hepatocyte growth factor (HGF) and insulin‐like growth factor 1 (IGF‐1) expression in ovarian tissue was observed by immunohistochemistry.ResultsOvarian function in lupus mice was abnormal, as indicated by decreased serum E2 and AMH concentrations, and increased FSH concentrations. HUC‐MSC transplantation caused significant upregulation of serum E2 and AMH and downregulation of FSH (all p &lt; 0.05). Ovarian structure improved and the follicle number increased after HUC‐MSC transplantation. Multiple infusions of HUC‐MSCs at Weeks 12 and 16 resulted in a significantly higher number of primordial follicles than infusions of HUC‐MSCs at only Week 12 (p &lt; 0.05). Immunohistochemistry showed that IGF‐1 and HGF expression increased after HUC‐MSC transplantation, but this was not significant.ConclusionsHUC‐MSCs transplantation restores disturbed hormone secretion and folliculogenesis in lupus mice. HUC‐MSC transplantation should be repeated for the best treatment effect.