Exosome-Mediated miR-21 Was Involved in the Promotion of Structural and Functional Recovery Effect Produced by Electroacupuncture in Sciatic Nerve Injury.

PURPOSE: Our study is aimed at investigating the mechanism by which electroacupuncture (EA) promoted nerve regeneration by regulating the release of exosomes and exosome-mediated miRNA-21 (miR-21) transmission. Furthermore, the effects of Schwann cells- (SC-) derived exosomes on the overexpression of miR-21 for the treatment of PNI were investigated. METHODS: A sciatic nerve injury model of rat was constructed, and the expression of miR-21 in serum exosomes and damaged local nerves was detected using RT-qPCR after EA treatment. The exosomes were identified under a transmission electron microscope and using western blotting analysis. Then, the exosome release inhibitor, GW4869, and the miR-21-5p-sponge used for the knockdown of miR-21 were used to clarify the effects of exosomal miR-21 on nerve regeneration promoted by EA. The nerve conduction velocity recovery rate, sciatic nerve function index, and wet weight ratio of gastrocnemius muscle were determined to evaluate sciatic nerve function recovery. SC proliferation and the level of neurotrophic factors were assessed using immunofluorescence staining, and the expression levels of SPRY2 and miR-21 were detected using RT-qPCR analysis. Subsequently, the transmission of exosomal miR-21 from SC to the axon was verified in vitro. Finally, the exosomes derived from the SC infected with the miR-21 overexpression lentivirus were collected and used to treat the rat SNI model to explore the therapeutic role of SC-derived exosomes overexpressing miR-21. RESULTS: We found that EA inhibited the release of serum exosomal miR-21 in a PNI model of rats during the early stage of PNI, while it promoted its release during later stages. EA enhanced the accumulation of miR-21 in the injured nerve and effectively promoted the recovery of nerve function after PNI. The treatment effect of EA was attenuated when the release of circulating exosomes was inhibited or when miR-21 was downregulated in local injury tissue via the miR-21-5p-sponge. Normal exosomes secreted by SC exhibited the ability to promote the recovery of nerve function, while the overexpression of miR-21 enhanced the effects of the exosomes. In addition, exosomal miR-21 secreted by SC could promote neurite outgrowth in vitro. CONCLUSION: Our results demonstrated the mechanism of EA on PNI from the perspective of exosome-mediated miR-21 transport and provided a theoretical basis for the use of exosomal miR-21 as a novel strategy for the treatment of PNI.

Schwann cells-derived exosomal miR-21 participates in high glucose regulation of neurite outgrowth.

As a common complication of diabetes, the pathogenesis of diabetic peripheral neuropathy (DPN) is closely related to high glucose but has not been clarified. Exosomes can mediate crosstalk between Schwann cells (SC) and neurons in the peripheral nerve. Herein, we found that miR-21 in serum exosomes from DPN rats was decreased. SC proliferation was inhibited, cell apoptosis was increased, and the expression of miR-21 in cells and exosomes was downregulated when cultured in high glucose. Increasing miR-21 expression reversed these changes, while knockdown of miR-21 led to the opposite results. When co-cultured with exosomes derived from SC exposed to high glucose, neurite outgrowth was inhibited. On the contrary, neurite outgrowth was accelerated when incubated with exosomes rich in miR-21. We further demonstrated that the SC-derived exosomal miR-21 participates in neurite outgrowth probably through the AKT signaling pathway. Thus, SC-derived exosomal miR-21 contributes to high glucose regulation of neurite outgrowth.

Low-temperature Heat Capacities and Thermodynamic Properties of Crystalline 2-Aminopyridinium Benzoate (C12H12N2O2) (s).

2-Aminopyridinium benzoate was synthesized. Chemical analysis, elemental analysis, and X-ray crystallography were applied to characterize the composition and crystal structure of the compound. The lattice potential energy of the title compound was calculated to be UPOT = 284.297 kJ mol-1. Low-temperature heat capacities of the compound were measured by a precision automatic adiabatic calorimeter over the temperature range from 78 K to 365 K. A polynomial equation of heat capacities against the temperature in the region of 78 K to 365 K was fitted by a least square method. Based on the fitted polynomial equation, the smoothed heat capacities and thermodynamic functions of the compound relative to the standard reference temperature 298.15 K were calculated at intervals of 5 K. According to the synthesis reaction, the standard molar enthalpies of dissolution for the reactants and product in the selected solvent were measured by an isoperibol solution-reaction calorimeter, respectively. Accordingly, the enthalpy change of the synthesis reaction was calculated to be ΔrHom = -(20.016 ± 0.182) kJ mol-1. Finally, the standard molar enthalpy of formation of 2-aminopyridinium benzoate was determined to be ΔfHom = - (365.416 ± 0.961) kJ mol-1 in accordance with Hess law.

Schwann cells apoptosis is induced by high glucose in diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus that affects approximately half of patients with diabetes. Current treatment regimens cannot treat DPN effectively. Schwann cells (SCs) are very sensitive to glucose concentration and insulin, and closely associated with the occurrence and development of type 1 diabetic mellitus (T1DM) and DPN. Apoptosis of SCs is induced by hyperglycemia and is involved in the pathogenesis of DPN. This review considers the pathological processes of SCs apoptosis under high glucose, which include the following: oxidative stress, inflammatory reactions, endoplasmic reticulum stress, autophagy, nitrification and signaling pathways (PI3K/AKT, ERK, PERK/Nrf2, and Wnt/ß-catenin). The clarification of mechanisms underlying SCs apoptosis induced by high glucose will help us to understand and identify more effective strategies for the treatment of T1DM DPN.

Electroacupuncture Promoted Nerve Repair After Peripheral Nerve Injury by Regulating miR-1b and Its Target Brain-Derived Neurotrophic Factor.

Growing evidence indicates that electroacupuncture (EA) has a definite effect on the treatment of peripheral nerve injury (PNI), but its mechanism is not completely clear. MicroRNAs (miRNAs) are involved in the regulation of a variety of biological processes, and EA may enhance PNI repair by regulating miRNAs. In this study, the rat sciatic nerve injury model was treated with EA for 4 weeks. Acupoints Huantiao (GB30) and Zusanli (ST36) were stimulated by EA 20 min once a day, 6 days a week for 4 weeks. We found that EA treatment downregulated the expression of miR-1b in the local injured nerve. In vitro experiments showed that overexpression of miR-1b inhibited the expression of brain-derived neurotrophic factor (BDNF) in rat Schwann cell (SC) line, while BDNF knockdown inhibited the proliferation, migration, and promoted apoptosis of SCs. Subsequently, the rat model of sciatic nerve injury was treated by EA treatment and injection of agomir-1b or antagomir-1b. The nerve conduction velocity ratio (NCV), sciatic functional index (SFI), and S100 immunofluorescence staining were examined and showed that compared with the model group, NCV, SFI, proliferation of SC, and expression of BDNF in the injured nerves of rats treated with EA or EA + anti-miR-1b were elevated, while EA + miR-1b was reduced, indicating that EA promoted sciatic nerve function recovery and SC proliferation through downregulating miR-1b. To summarize, EA may promote the proliferation, migration of SC, and nerve repair after PNI by regulating miR-1b, which targets BDNF.

[Isolation, purification and primary culture of rat pancreatic beta-cells].

OBJECTIVE: To isolate and purify rat pancreatic beta-cells and to explore the best conditions for the primary culture of the pancreatic beta-cells in vitro. METHODS: The pancreas of Norman Wistar rats were digested by collagenase V. The islets were purified by mesh sieve. The activity of the islets was stimulated by different concentrations of glucose and detected by dithizone dye. The purified islets were put into RPMI-1640 nutritive medium for culture overnight. The cultured islets were digested again with trypsin and DNAase to obtain the suspension containing single pancreatic cells. The beta-cells were separated and purified in a fluorescence-activated cell sorter (FACS) in the medium containing 2.8 mmol/L glucose. The purified beta-cells were identified by immunohistochemistry and glucose stimulating test. Ham's F-10 with different concentrations of glucose and 3-Isobutyl-1-methylxanthine (IBMX) were used as nutritive medium for the primary cell culture for 24 hours. The best conditions for the culture were identified. RESULTS: An average of 550 +/- 90 islets with fine activities were obtained per rat. The purification with FACS obtained about 5688 beta-cells per rat, with a recovery rate of (93.69 +/- 1.26)% and a purity of (85.5 +/- 1.24)%. A concentration of 10.0 mmol/L and 16.0 mmol/L glucose in primary culture for 24 hours produced the highest survival rates of beta-cells, but IBMX did not increase the survival rates of beta-cells. CONCLUSION: FACS is effective in purifying pancreatic beta-cells from the suspension with a medium containing 2.8 mmol/L glucose. Pancreatic beta-cells maintain relatively high activities in Ham's F-10 medium containing 10.0-16.0 mmol/L glucose in primary culture.

miR-1b overexpression suppressed proliferation and migration of RSC96 and increased cell apoptosis.

Peripheral nerve injury (PNI) is a global problem that leads to severe disability and high healthcare expenditure. Accumulating evidence suggested that the phenotypes of Schwann cells (SCs) could be regulated by microRNAs (miRNAs) and expressions of various miRNAs are altered after PNI. In this study, the expression of miR-1b in the injured nerve and hypoxia-treated SCs was detected through qRT-PCR. The target genes of miR-1b were predicted by bioinformatics prediction and dual-luciferase reporter assay and verified through qRT-PCR and western blot. The effects of miR-1b and its specific target gene on the proliferation, migration and apoptosis of SCs were determined and the regulation of miR-1b on peripheral nerve regeneration after PNI was further investigated in vivo. We found that miR-1b was obviously downregulated in the injured nerve in a rat sciatic nerve transection model and directly targeted N-myc downstream-regulated gene 3 (NDRG3) by binding to its 3'-UTR and caused both mRNA degradation and translation suppression of NDRG3. Overexpression of miR-1b or knockdown of NDRG3 decreased the proliferation and migration as well as increased the apoptosis of SCs. NDRG3 reversed the effects of miR-1b overexpression on proliferation/migration/apoptosis of RSC96. In addition, injection of miR-1b antagomir promoted the expression of NDRG3 in the injured nerve following sciatic nerve injury. Compared to the model group, the rats treated with miR-1b agomir had lower functional recovery rate, and downregulation of miR-1b through injection of specific antagomir improved the functional recovery rate according to the results of sciatic functional index and nerve conduction velocity. Overall, our results will contribute to the development of novel targets for promoting nerve regeneration after PNI.

Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion.

Using electroacupuncture and moxibustion to treat peripheral nerve injury is highly efficient with low side effects. However, the electroacupuncture- and moxibustion-based mechanisms underlying nerve repair are still unclear. Here, in vivo and in vitro experiments uncovered one mechanism through which electroacupuncture and moxibustion affect regeneration after peripheral nerve injury. We first established rat models of sciatic nerve injury using neurotomy. Rats were treated with electroacupuncture or moxibustion at acupoints Huantiao (GB30) and Zusanli (ST36). Each treatment lasted 15 minutes, and treatments were given six times a week for 4 consecutive weeks. Behavioral testing was used to determine the sciatic functional index. We used electrophysiological detection to measure sciatic nerve conduction velocity and performed hematoxylin-eosin staining to determine any changes in the gastrocnemius muscle. We used immunohistochemistry to observe changes in the expression of S100-a specific marker for Schwann cells-and an enzyme-linked immunosorbent assay to detect serum level of nerve growth factor. Results showed that compared with the model-only group, sciatic functional index, recovery rate of conduction velocity, diameter recovery of the gastrocnemius muscle fibers, number of S100-immunoreactive cells, and level of nerve growth factor were greater in the electroacupuncture and moxibustion groups. The efficacy did not differ between treatment groups. The serum from treated rats was collected and used to stimulate Schwann cells cultured in vitro. Results showed that the viability of Schwann cells was much higher in the treatment groups than in the model group at 3 and 5 days after treatment. These findings indicate that electroacupuncture and moxibustion promoted nerve regeneration and functional recovery; its mechanism might be associated with the enhancement of Schwann cell proliferation and upregulation of nerve growth factor.

[The effect of C-SiO2 composite films on corrosion resistance of dental Co-Cr alloy].

PURPOSE: To study the effect of carbon-silica composite films on corrosion resistance of Co-Cr alloy in simulated oral environment and provide evidences for clinical application of this new material. METHODS: Co-Cr alloy specimens were cut into appropriate size of 20 mm × 20 mm × 0.5 mm. Then, the carbon-silica composite films were spin-coated onto the specimens. Subsequently, ICP-AES was used to observe the Co, Cr, Mo ion concentrations. Finally, Tafel polarization curves of the specimens were used to measure the electrochemical corrosion resistance by electrochemical workstation. SAS8.0 software package was used for statistical analysis. RESULTS: The results of ICP-AES showed that the ion concentrations of Co, Cr, Mo of specimens coated with composite films in the testing liquid were significantly smaller than that of Co-Cr alloy specimens. Tafel polarization curves showed that in the specimens coated with composite films, the corrosion potential moved in the positive direction and increased from -0.261 V to -0.13 V. At the same time, the corrosion current density decreased from -5.0017µA/cm2 to -5.3006 µA/cm2. CONCLUSIONS: Carbon-silica composite films (silica=61.71wt %) can reduce the release of metal ions significantly and improve the corrosion resistance of Co-Cr alloys effectively. Carbon-silica composite films may be a promising dental material.