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Suture and autologous nerve transplantation are the primary therapeutic measures for completely severed nerves. However, imbalances in the microenvironment and adhesion of surrounding tissues can affect the quality of nerve regeneration and repair. Previous studies have shown that human amniotic membrane can promote the healing of a variety of tissues. In this study, the right common peroneal nerve underwent a 5-mm transection in rats. Epineural nerve repair was performed using 10/0 non-absorbable surgical suture. The repair site was wrapped with a two-layer amniotic membrane with α-cyanoacrylate rapid medical adhesive after suture. Hindlimb motor function was assessed using footprint analysis. Conduction velocity of the common peroneal nerve was calculated by neural electrical stimulation. The retrograde axoplasmic transport of the common peroneal nerve was observed using fast blue BB salt retrograde fluorescent staining. Hematoxylin-eosin staining was used to detect the pathological changes of the common peroneal nerve sputum. The mRNA expression of axon regeneration-related neurotrophic factors and inhibitors was measured using real-time polymerase chain reaction. The results showed that the amniotic membrane significantly improved the function of the injured nerve; the toe spread function rapidly recovered, the nerve conduction velocity was restored, and the number of fast blue BB salt particles were increased in the spinal cord. The amniotic membrane also increased the recovery rate of the tibialis anterior muscle and improved the tissue structure of the muscle. Meanwhile, mRNA expression of nerve growth factor, growth associated protein-43, collapsin response mediator protein-2, and brain-derived neurotrophic factor recovered to near-normal levels, while Lingo-1 mRNA expression decreased significantly in spinal cord tissues. mRNA expression of glial-derived neurotrophic factor did not change significantly. Changes in mRNA levels were more significant in amniotic-membrane-wrapping-treated rats compared with model and nerve sutured rats. These results demonstrate that fresh amniotic membrane wrapping can promote the functional recovery of sutured common peroneal nerve via regulation of expression levels of neurotrophic factors and inhibitors associated with axonal regeneration. The study was approved by the Committee on Animal Research and Ethics at the Affiliate Hospital of Zunyi Medical University, China (approval No. 112) on December 1, 2017.

It has been reported that Neonatal hypoxic-ischemic encephalopathy (HIE) could induce apoptosis in neonates and result in cognitive and sensory impairments, which are associated with poor developmental outcomes. Despite the improvement in neonatology, there is still no clinically effective treatment for HIE presently. Long non-coding RNAs (lncRNAs) play important roles in cellular homeostasis. Nevertheless, their effects in developing rat brains with HI is little known. Here, we established HIE model in neonate rats and explored the expression and function of lncRNAs in HI, and found the expression of 19 lncRNAs was remarkably changed in the brains of HI rats, compared to the sham group. Among them, three lncRNAs (TCONS_00041002, TCONS_00070547, TCONS_00045572) were enriched in the apoptotic process via gene ontology (GO) and pathway analysis, which were selected for the further qRT-PCR verification. Through lentivirus-mediated overexpression of these three lncRNAs, we found that overexpression of TCONS_00041002 attenuated the cell apoptosis, and increased the vitality of neurons after oxygen-glucose deprivation (OGD), therefore reduced the brain infarction and further promoted the neuron survival as well as improved the neurological disorders in the rats subjected to HIE. What's more, ceRNA network prediction and co-expression verification showed that the expression of TCONS_00041002 was positively associated with Foxe1, Pawr and Nfkbiz. Altogether, this study has exhibited that lncRNA TCONS_00041002 participates in the cell apoptosis and neuronal survival of HIE and represents a potential new target for the treatment of HIE. Refereed/Peer-reviewed

CD44 antigen (CD44) is a transmembrane protein found in cell adhesion molecules and is involved in the regulation of various physiological processes in cells. It was hypothesized that CD44 directly affected the chondrogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs). In the present study, the expression of chondrocyte-associated factors was detected in the absence and presence of the antibody blocker anti-CD44 antibody during the chondrogenic differentiation of hAMSCs. Following inhibition of CD44 expression, the transcriptional levels of chondrocyte-associated genes SRY-box transcription factor 9, aggrecan and collagen type II α 1 chain, as well as the production of chondrocyte markers type II collagen and aggrecan were significantly decreased in hAMSCs. Further investigation indicated that there was no significant change in total ERK1/2 expression following inhibition of CD44 expression; however, phosphorylated (p)-ERK1/2 expression was decreased. The expression of p-Smad2/3 was also upregulated following CD44 inhibition. These data indicated that CD44 may affect the differentiation of hAMSCs into chondrocytes by regulating the Smad2/3 and ERK1/2 signaling pathway.

Osteogenic inducers play central roles in effective stem cell-based treatment of bone defects/losses. However, the current routine osteogenic inducer is a cocktail comprising three components that must be improved due to low induction efficiency and side effects. Therefore, there is an urgent need to develop safer and more effective osteoinducers. Herein, we demonstrated the osteogenic effect of Ganoderal A (GD-A), a tetracyclic triterpenoid compound from Ganoderma lucidum. GD-A showed no cytotoxicity toward human amniotic mesenchymal stem cells (hAMSCs) at doses of 0.001-10 μM; furthermore, 0.01 μM GD-A significantly induced the generation of osteoblast-specific markers, such as alkaline phosphatase, and calcium deposition in hAMSCs. At molecular levels, GD-A promoted the expression of multiple osteoblast differentiation markers, such as RUNX2, OSX, OPN, ALP, OCN, and COL1α1. Both Wnt/β-catenin and BMP/SMAD signaling were shown as active during hAMSC osteodifferentiation. Furthermore, specific blocking of both signals by KYA1797K and SB431542 significantly inhibited alkaline phosphatase secretion and RUNX2 and ALP expression when used alone or in combination. Meanwhile, both signals were also blocked. These findings suggest that GD-A induces hAMSC differentiation into osteoblasts through signaling cross-talk between Wnt/β-catenin and BMP/SMAD. Taken together, GD-A is a safe, effective, and novel osteoinducer and might be used for stem cell-based therapy for bone defects/losses.

Neonatal hypoxic-ischemic encephalopathy (HIE) is major cause of neonatal death or long-term neurodevelopmental disabilities, which becomes a major practical problem currently in clinic. Whereas, its pathophysiology and underlying molecular mechanism is not clear. MicroRNAs are involved in the normal growth and development of neuronal cells. Herein, the objective of this research was to examine the roles of miR-410-3p in neurological deficits, neuronal injury and neuron apoptosis after hypoxic-ischemic and to explore its associated mechanisms. We established the hypoxic-ischemic brain damage (HIBD) model and oxygen glucose deprivation (OGD) model. Zea-longa score and TTC staining were used to detect the acute cerebral dysfunction after HIBD. QPCR verification exhibited notable downregulation of miR-410-3p expression at 24 h in rats after HIBD as well as that in PC12, SY5Y cells and primary cortical neurons post OGD. To further determine the function of miR-410-3p, lentivirus-mediated overexpression virus was applied in vivo and in vitro. Behavioral tests, including Morris water maze, open field test, Y maze test, neurological severity score and rotating rod test, were performed to evaluate long-term behavioral changes of rats at 1 month post HIBD. The results showed that the number of cells together with the axonal length were reduced post OGD. While the increase of cells number and the axonal length was measured after upregulating miR-410-3p. Meanwhile, miR-410-3p overexpression inhibited neuron apoptosis and enhanced neuronal survival. In addition, long-term motor and cognitive functions were remarkably recovered in HIBD rats with miR-410-3p overexpression. Together, miR-410-3p exerts a critical role in protecting neuronal growth as well as promoting motor and cognitive function recovery in neonatal rats subjected to HIBD. The current study therefore provides critical insights to develop the activator of miR-410-3p for the clinical treatment of HIBD in future clinic trial Refereed/Peer-reviewed

Osteoarthritis (OA) is one of the most common refractory degenerative articular cartilage diseases. Human amniotic mesenchymal cells (hAMSCs) have emerged as a promising stem cell source for cartilage repair, and hyaluronic acid (HA) has proven to be a versatile regulator for stem cell transplantation. Herein, an effective and straightforward intra-articular injection therapy using a cocktail of hAMSCs and HA was developed to treat knee OA in a rat model. The injured cartilage was remarkably regenerated, yielding results comparable to normal cartilage levels after 56 days of treatment. Both hAMSCs and HA were indispensable organic components in this therapy, in which HA could synergistically enhance the effects of hAMSCs on cartilage repair. The regenerative mechanism was attributed to the fact that the addition of HA comprehensively enhances the activities of hAMSCs, including chondrogenic differentiation, proliferation, colonization, and regenerative modulation. This cocktail paves a new avenue for injection therapy to treat OA, holding the potential to realize rapid clinical translation.

Aims This study investigated the effects of hyaluronic acid (HA), a commonly used osteogenic medium referred to as DAG, and the combined administration of HA and DAG (CG) on the osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs), and the underlying mechanism. Main methods The phenotype of hAMSCs was detected by flow cytometry and immunocytochemical staining. Alkaline phosphatase (ALP) and calcium deposition assays were employed for evaluating the osteogenic differentiation of hAMSCs. The expression of osteogenesis-related genes and proteins was determined by quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Meanwhile, the molecular mechanism of osteogenic differentiation of hAMSCs was detected by PCR array and qRT-PCR. Key findings The results showed that treatment with CG could significantly stimulate hAMSC ALP activity and calcium deposition compared to treatment with DAG, while HA had little effect. The expression of osteogenesis-related molecules and stemness-related molecules was up-regulated at the mRNA and protein levels in all three groups, and this up-regulation was most significant in the CG group. In addition, treatment with CG significantly increased the gene expressions involved in regulation of the TGF-β/Smad signalling pathway compared to treatment with DAG. Furthermore, the pro-osteogenic differentiation effects as well as the up-regulated expression of genes observed in the CG treatment group were significantly inhibited when the cells were pre-treated with SB431542, an inhibitor of the TGF-β/Smad pathway. Significance These results suggest that HA in combination with DAG could significantly enhance the osteogenic differentiation of hAMSCs, potentially via the TGF-β/Smad signalling pathway.

Our hypothesis is that hyaluronic acid may regulate the differentiation of human amniotic epithelial cells (hAECs) into insulin-producing cells and help the treatment of type 1 diabetes. Herein, a protocol for the stepwise in vitro differentiation of hAECs into functional insulin-producing cells was developed by mimicking the process of pancreas development. Treatment of hAECs with hyaluronic acid enhanced their differentiation of definitive endoderm and pancreatic progenitors. Endodermal markers Sox17 and Foxa2 and pancreatic progenitor markers Pax6, Nkx6.1, and Ngn3 were upregulated an enhanced gene expression in hAECs, but hAECs did not express the β cell-specific transcription factor Pdx1. Interestingly, hyaluronic acid promoted the expression of major pancreatic development-related genes and proteins after combining with commonly used inducers of stem cells differentiation into insulin-producing cells. This indicated the potent synergistic effects of the combination on hAECs differentiation in vitro. By establishing a multiple injection transplantation strategy via tail vein injections, hAECs transplantation significantly reduced hyperglycemia symptoms, increased the plasma insulin content, and partially repaired the islet structure in type 1 diabetic mice. In particular, the combination of hAECs with hyaluronic acid exhibited a remarkable therapeutic effect compared to both the insulin group and the hAECs alone group. The hAECs' paracrine action and hyaluronic acid co-regulated the local immune response, improved the inflammatory microenvironment in the damaged pancreas of type 1 diabetic mice, and promoted the trans-differentiation of pancreatic α cells into β cells. These findings suggest that hyaluronic acid is an efficient co-inducer of the differentiation of hAECs into functional insulin-producing cells, and hAECs treatment with hyaluronic acid may be a promising cell-replacement therapeutic approach for the treatment of type 1 diabetes.

BackgroundManganese (Mn) is widely used in industries including the manufacture of Mn-iron (Fe) alloy. Occupational Mn overexposure causes manganism. Mn is known to affect Fe metabolism; this study was designed to test the hypothesis that workers exposed to Mn may have an altered expression of mRNAs encoding proteins in Fe metabolism.MethodsWorkers occupationally exposed to Mn (n= 71) from a Mn–Fe alloy factory and control workers without Mn-exposure (n= 48) from a pig-iron plant from Zunyi, China, were recruited for this study. Blood samples were collected into Trizol-containing tubes. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. Metal concentrations were quantified by atomic absorption spectrophotometry.ResultsWorking environment and genetic background of both groups were similar except for marked differences in airborne Mn concentrations (0.18 mg/m3in Mn–Fe alloy factory vs. 0.0022 mg/m3in pig-Fe plant), and in blood Mn levels (34.3 µg/L vs. 10.4 µg/L). Mn exposure caused a significant decrease in the expression of divalent metal transporter-1 (DMT1), transferrin (Tf) and hepcidin by 58.2%, 68.5% and 61.5%, respectively, as compared to controls, while the expression of transferrin receptor (TfR) was unaltered. Linear regression analysis revealed that expressions of DMT1, Tf and hepcidin were inversely correlated with the accumulative Mn exposure; the correlation coefficients (r) are −0.47, −0.54, and −0.49, respectively (p< 0.01).ConclusionThe data suggest that occupational Mn exposure causes decreased expressions of DMT1, Tf and hepcidin in blood cells; the finding will help understand the mechanism underlying Mn exposure-associated alteration in Fe homeostasis among workers.