RSC96 Cells Research Articles

IFN-γ/mTORC1 decreased Rab11 in Schwann cells of diabetic peripheral neuropathy, inhibiting cell proliferation via GLUT1 downregulation.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus. Rab11 is conserved gene-regulating vesicle traffic and reported to be involved in the pathogenesis of diabetes mellitus by affecting insulin sensitivity. We aimed to investigate the role of Rab11 in the pathogenesis of DPN. In this study, Rab11 expression decreased in the sciatic nerves of diabetic mice with impaired conduction function versus those of normal mice. In vitro experiment revealed interferon-γ (IFN-γ), not high glucose and interleukin 1β was the main factor to lead to Rab11 downregulation in RSC96 cells. Again, both Rab11 knockdown and IFN-γ treatment caused cell viability inhibition and the decrease in BrdU-positive cells. In contrast, overexpression of Rab11 reversed IFN-γ-reduced cell proliferation. Furthermore, mTORC1 not mTORC2 was proven to be suppressed by IFN-γ treatment in RSC96 cells, indicated in decreased phospho-p70S6K. Inhibition of the mTORC1 pathway resulted in Rab11 expression downregulation in RSC96 cells. Activation of the mTORC1 pathway effectively prevented IFN-γ-reduced Rab11 expression in RSC96 cells. Also, glucose transporter 1 (GLUT1) was found to be downregulated in RSC96 cells with Rab11 silence and overexpression of GLUT1 reversed Rab11 blocking-caused proliferation inhibition. Taken together, our findings suggest that IFN-γ decreases Rab11 expression via the inhibition of the mTORC1 signaling pathway, causing reduced cell proliferation in Schwann cells of DPN by GLUT1 downregulation.

Activation of AMPK pathway compromises Rab11 downregulation-mediated inhibition of Schwann cell proliferation in a Glut1 and Glut3-dependent manner

Rab11, a small GTPase, is an important protein in the regulation of intracellular plasma membrane trafficking. Schwann cells are the main cells of peripheral nerves and knockdown of Rab11 in these cells inhibits the formation of functional tunneling nanotubes (TNTs). However, the role of Rab11 in the functioning of Schwann cells remains elusive. Herein, using cell viability analysis, live/dead cell staining, BrdU assay, and western blot analysis with an AMPK antibody, we observed that the knockdown of Rab11 significantly inhibited the proliferation of RSC96 cells. Further investigations showed that the AMPK pathway was activated by the knockdown of Rab11, as indicated by the enhanced levels of phosphorylated AMPK. Moreover, suppression of AMPK pathway with Compound C aggravated Rab11 knockdown-induced inhibition of cell proliferation. In contrast, activation of the AMPK pathway with AICAR ameliorated the Rab11 knockdown-mediated inhibition of cell proliferation. Furthermore, the levels of Glut1 and Glut3 were decreased in the RSC96 cells upon Rab11 knockdown. Additionally, the knockdown of Glut1 and Glut3 led to the activation of the AMPK pathway in RSC96 cells. We conclude that the knockdown of Rab11 suppresses the proliferation of RSC96 cells, and as a compensatory mechanism, the activation of AMPK pathway, in a Glut1 and Glut3-dependent manner, improves RSC96 cell proliferation.

Notoginsenoside R1 alleviates high glucose-evoked damage in RSC96 cells through down-regulation of miR-503

Notoginsenoside R1 (NGR1) is a major bioactive ingredient of Radix notoginseng that has promise in treating several diabetes-related diseases, like diabetic nephropathy and diabetic cardiomyopathy. This work is designed to explore if NGR1 has potential in treating diabetic peripheral neuropathy (DPN). A cell model of DPN was made by stimulating RSC96 cells with high glucose. The effects of NGR1 preconditioning were examined by conducting CCK-8 assay, flow cytometry, ROS assay and Western blot. The downstream effector and signalling of NGR1 were then explored by qRT-PCR and Western blot. High glucose incubation led to cell viability loss, apoptosis facilitation, caspase-3 and PARP cleavage, and ROS generation of RSC96 cells. Meanwhile, expression of NGF and BDNF was declined by high glucose. Preconditioning NGR1 significantly protected RSC96 cells against high glucose-evoked damage. And NGR1 prevented high glucose-induced expression of miR-503. The beneficial functions of NGR1 towards high glucose-injured RSC96 cells were impeded by miR-503 overexpression. Further, NGR1 activated PI3K/AKT and β-catenin signalling through a miR-503-dependent way. This paper illustrated the neuroprotective and neurotrophic function of NGR1 in RSC96 cells. The beneficial function may due to its regulation on miR-503 expression and the downstream signalling such as PI3K/AKT and β-catenin.

Taurine protects against myelin damage of sciatic nerve in diabetic peripheral neuropathy rats by controlling apoptosis of schwann cells via NGF/Akt/GSK3β pathway

Diabetic peripheral neuropathy is a common complications of Type 2 Diabetes and its main pathological feature is myelin sheath damage of peripheral nerve that was induced by Schwann cells (SCs) apoptosis. Increasing evidence suggested that taurine might play a role in improving DPN because of its ability to prevent SCs apoptosis. In this study, we explore the effect of taurine on preventing SCs apoptosis and its underlying mechanism. Sprague Dawley rats were treated with streptozotocin to establish the diabetes model. Rats were randomly divided into control, diabetes, taurine treatment (as giving 0.5%, 1% and 2% taurine in drinking water) groups. RSC96 cell (a rat SCs line) was used for intervention experiments in vitro. Results showed that taurine significantly corrected morphology of damaged myelin sheath and inhibited SCs apoptosis in sciatic nerve of diabetic rats. Moreover, taurine prevented apoptosis of RSC96 cells exposed to high glucose. Mechanistically, taurine up-regulated NGF expression and phosphorylation levels of Akt and GSK3β, while, blocking activation of NGF and phosphorylation of Akt and GSK3β increased apoptosis of high glucose-exposed RSC96 cells with taurine supplement. These results revealed taurine improved the myelin sheath damage of sciatic nerve in diabetic rats by controlling SCs apoptosis via NGF/Akt/GSK3β signaling pathways, which provides some clues that taurine might be effective and feasible candidate for the treatment of DPN.

The Akt/mTOR cascade mediates high glucose-induced reductions in BDNF via DNMT1 in Schwann cells in diabetic peripheral neuropathy

Brain-derived neurotropic factor (BDNF) deficiency in Schwann cells plays an important role in the pathogenesis of diabetic peripheral neuropathy (DPN). Little is known about the mechanism involved in BDNF downregulation in Schwann cells in DPN. In this study, we first confirmed downregulation of BDNF and neurotrophin 3 expression in the sciatic nerves of diabetic mice, which was accompanied by myelin sheath abnormalities. Moreover, in vitro, high glucose was revealed to cause downregulation of BDNF, but not neurotrophin 3, expression in RSC96 cells, which was accompanied by DNA hypermethylation of BDNF promoters I and II. DNMT1 was subsequently revealed to be enhanced at the mRNA and protein levels in high glucose-stimulated RSC96 cells, and inhibition of DNMT1 with 5-Aza treatment or shRNA vector transfection reversed high glucose-induced reductions in BDNF expression. Furthermore, the mTOR and upstream Akt pathways were indicated to mediate high glucose-induced DNMT1 and BDNF expression in RSC96 cells. Taken together, our results suggest that the Akt/mTOR cascade mediates high glucose-induced reductions in BDNF via DNMT1 in Schwann cells in DPN.

Reciprocal interactions between cancer and Schwann cells contribute to oral cancer progression and pain

Pain associated with oral squamous cell carcinoma (oral SCC) decreases quality of life and survival. The interaction between cancer and the peripheral nerves is known to initiate and amplify pain and contribute to carcinogenesis. Schwann cells envelop peripheral nerves and are activated in response to neuronal damage. The contributions of Schwann cells to oral SCC progression and pain are unknown. Using a non-contact co-culture model, we demonstrate that Schwann cells (RSC-96) and oral SCC cells (HSC-3) reciprocally interact to promote proliferation, migration, and invasion. Schwann cell-oral SCC interaction leads to increased production of adenosine, which stimulates cell proliferation and migration of both cell types. The adenosine receptor A2B (ADORA2B) is expressed on RSC-96 cells. We show that supernatant from the RSC-96 cells co-cultured with HSC-3 cells induces increased mechanical hypersensitivity in mice compared to supernatant from control RSC-96 cells. Treatment with the ADORA2B antagonist PSB603 significantly inhibits co-culture interactions - proliferation and migration, and co-culture supernatant induced mechanical hypersensitivity. RSC-96 cells co-cultured with HSC-3 cells secrete increased amounts of the pronociceptive mediator, interleukin-6 (IL-6), which can be reduced by adding PSB603 into the co-culture. Our data support a reciprocal interaction between oral SCC and Schwann cells mediated by adenosine with potential to promote oral SCC progression and pain via increased secretion of IL-6.

Anti-apoptotic Effect of Taurine on Schwann Cells Exposed to High Glucose In Vitro.

It was reported that apoptosis of Schwann cells could increase in the diabetic rats. The studies showed that taurine inhibited apoptosis in a variety of cells. However, there were few reports on studying the protection of taurine against apoptosis of Schwann cells induced by high glucose (HG) and the underlying mechanism. In our study, the cells were divided into five groups: Control: the normal medium; HG group: 50mM high glucose; T1: 50mM high glucose+Taurine (10mM) group; T2: 50mM high glucose+Taurine (20mM) group; T3: 50mM high glucose+Taurine (40mM) group. We used MTT and Tunel assays to measure the cell viability and apoptosis, respectively. Then, we also used western blotting to detect the protein levels of apoptosis-related protein. The results demonstrate that taurine promoted cell viability and decreased apoptosis in RSC96 cells exposed to HG. Furthermore, taurine markedly improved imbalance of Bax and Bcl-2, inhibited the translocation of Cytochrome C (Cyt C) from mitochondria to cytosol and reduced caspase-3 activity in HG-induced RSC96 cells. Our results indicate that taurine protect against apoptosis of Schwann cells induced by HG via inhibiting mitochondria-dependent caspase-3 pathway.

Salvianolic acid B inhibits the development of diabetic peripheral neuropathy by suppressing autophagy and apoptosis.

The aim of this study was to evaluate the neuroprotective effects of SalB on high glucose (HG)-induced excessive autophagy and apoptosis invitro. The proliferation and apoptosis of RSC96 cells were determined using the MTT assay and flow cytometry, respectively. Western blot analysis was performed to examine the expression of autophagy and apoptosis-related proteins. RT-PCR and flow cytometry were manipulated to examine the level of Bcl-2. The signals of autophagy markers were detected using immunofluorescence methods. We found that HG significantly reduced RSC96 cell's proliferation and induced apoptosis. What's more, HG increased the level of autophagy and apoptosis-related proteins. However, these effects were reversed by SalB. In addition, we also found that 3-MA decreased the expression of LC3A/B and Beclin1, while the JNK inhibitor SP600125 reduced the levels of phosphorylated JNK, LC3A/B and Beclin1. High glucose not only induced apoptosis but also caused autophagic cell death by activating the JNK pathway. These effects prevented by SalB in an opposite manner.

Improving the protective effects of aFGF for peripheral nerve injury repair using sulfated chitooligosaccharides

Injury to the peripheral nerves can result in temporary or life-long neuronal dysfunction and subsequent economic or social disability. Acidic fibroblast growth factor (aFGF) promotes the growth and survival of neurons and is a possible treatment for peripheral nerve injury. Yet, the actual therapeutic utility of aFGF is limited by its short half-life and instability in vivo. In the present study, we prepared sulfated chitooligosaccharides (SCOS), which have heparin-like properties, to improve the bioactivity of aFGF. We investigated the protective effects of SCOS with or without aFGF on RSC96 cells exposed to Na2S2O4 hypoxia/reoxygenation injury. Cell viability was measured by MTT assay and cytotoxicity induced by Na2S2O4 was assessed by lactate dehydrogenase (LDH) release into the culture medium. Pretreatment with aFGF and SCOS dramatically decreased LDH release after injury compared to pretreatment with aFGF or SCOS alone. We subsequently prepared an aFGF/SCOS thermo-sensitive hydrogel with poloxamer and examined its effects in vivo. Paw withdrawal thresholds and thermal withdrawal latencies were measured in rats with sciatic nerve injury. Local injection of the aFGF/SCOS hydrogels (aFGF: 40, 80 µg/kg) increased the efficiency of sciatic nerve repair compared to aFGF (80 µg/kg) hydrogel alone. Especially aFGF/SCOS thermo-sensitive hydrogel decreased paw withdrawal thresholds from 117.75 ± 8.38 (g, 4 d) to 65.74 ± 3.39 (g, 10 d), but aFGF alone group were 140.58 ± 27.54 (g, 4 d) to 89.12 ± 5.60 (g, 10 d) (aFGF dose was 80 µg/kg, P < 0.05, n = 8). The thermal withdrawal latencies decreased from 11.61 ± 2.26 (s, 4 d) to 2.37 ±0.67 (s, 10 d). However, aFGF alone group were from 17.69 ± 1.47 (s, 4 d) to 4.65 ± 1.73 (s, 10 d) (P < 0.05, n = 8). Furthermore, the aFGF/SCOS hydrogels also exhibited good biocompatibility in mice. In summary, SCOS improved the protective effects of aFGF in RSC96 cells injured with Na2S2O4 and increased the efficiency of nerve repair and recovery of function in rats with sciatic nerve injury. These findings pave an avenue for the development of novel prophylactic and therapeutic strategies for peripheral nerve injury.

Prolonged high‐glucose exposure decreased SREBP‐1/FASN/ACC in Schwann cells of diabetic mice via blocking PI3K/Akt pathway

Abnormal lipid metabolism and SREBP-1 downregulation are reported to be involved in the pathogenesis of diabetic peripheral neuropathy (DPN). In the current study, the relationship between PI3K/Akt signaling pathway and SREBP-1 expression was explored in Schwann cells of DPN. The phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression were inhibited in the sciatic nerves of diabetic mice versus those of normal mice, accompanied with the atrophy of nerve fiber and the irregular myelin sheath. High concentration glucose suppressed phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression in cultured Schwann cell (RSC96 cell) in vitro, and 25 mmol/L glucose was enough to lead to the maximum inhibitory effect. The time-course effect of high glucose showed that Akt phosphorylation gradually decreased with the extension of stimulation time. Somewhat differently, short-term high-glucose exposure enhanced SREBP-1 expression and prolonged high-glucose stimulation reduced the SREBP-1 expression in RSC96 cells. Similarly, prolonged high-glucose stimulation also downregulated FASN messenger RNA (mRNA), ACC mRNA, intracellular triglyceride, and cholesterol. LY294002 suppressed Akt activation followed by the decreased SREBP-1, FASN, ACC, triglyceride, and cholesterol. Contrarily, the PI3K/Akt signaling pathway agonist insulin pretreatment avoided prolonged high-glucose stimulation-blocked Akt activation and increased SREBP-1, FASN, and ACC expression in the levels of protein and mRNA in RSC96 cells. The knockdown of SREBP-1 by shRNA prevented insulin-induced enhanced FASN, ACC mRNA expression, triglyceride, and cholesterol in high-glucose-treated RSC96 cells. In conclusion, prolonged high-glucose exposure inhibits the SREBP-1/FASN/ACC expression in the Schwann cells of DPN via the blockage of the PI3K/Akt signaling pathway.

DNMT1, a Novel Regulator Mediating mTORC1/mTORC2 Pathway-Induced NGF Expression in Schwann Cells

Schwann cells play an important role in maintaining the normal function of peripheral nerves via the secretion of nerve growth factor (NGF). The mTOR signaling pathway is known as a kind of Ser/Thr protein kinase that regulates various cell functions. DNA methyltransferase 1 (DNMT1) is an epigenetic regulator and downstream target of the mTOR pathway. In the present study, we explored the relationship between NGF expression and the mTOR pathway/DNMT1 in RSC96 cells. The results showed that both rapamycin and Torin 1 downregulated NGF expression via the inhibition of phospho-mTOR (Ser 2448) and phospho-S6K1 (Thr 389). Similarly, the silencing of RAPTOR and RICTOR decreased NGF expression by 56.7% and 52.4%, respectively, in RSC96 cells compared with the control siRNA treatment, which was accompanied by reduced phospho-S6K1 (Thr 389). The mTOR/S6K1 activator MHY1485 increased NGF expression by 28.7% and 17.1% 1day and 2day after stimulation, respectively, compared to the corresponding control group in RSC96 cells. Furthermore, DNMT1 was enhanced by 94.5% and 42.5% with mTOR pathway inhibitor (rapamycin and Torin 1, respectively) treatment for 3day compared with the control group. Additionally, the inhibition of DNMT1 with a chemical inhibitor or a specific shRNA plasmid upregulated NGF in RSC96 cells. In summary, our findings suggest that DNMT1 is the downstream target of the mTOR pathway and mediates the mTOR pathway inhibition-induced reduction in NGF expression in Schwann cells. Activation of the mTOR signaling pathway and/or inhibition of DNMT1 increased NGF expression, which may benefit patients suffering from NGF deficiencies, such as diabetic peripheral neuropathy.

Morphology, Migration, and Transcriptome Analysis of Schwann Cell Culture on Butterfly Wings with Different Surface Architectures.

It has been shown that material surface topography greatly affects cell attachment, growth, proliferation, and differentiation. However, the underlying molecular mechanisms for cell-material interactions are still not understood well. Here, two kinds of butterfly wings with different surface architectures were employed for addressing such an issue. Papilio ulysses telegonus (P.u.t.) butterfly wing surface is composed of micro/nanoconcaves, whereas Morpho menelaus (M.m.) butterfly wings are decorated with grooves. RSC96 cells grown on M.m. wings showed a regular sorting pattern along with the grooves. On the contrary, the cells seeded on P.u.t. wings exhibited random arrangement. Transcriptome sequencing and bioinformatics analysis revealed that huntingtin (Htt)-regulated lysosome activity is a potential key factor for determining cell growth behavior on M.m. butterfly wings. Gene silence further confirmed this notion. In vivo experiments showed that the silicone tubes fabricated with M.m. wings markedly facilitate rat sciatic nerve regeneration after injury. Lysosome activity and Htt expression were greatly increased in the M.m. wing-fabricated graft-bridged nerves. Collectively, our data provide a theoretical basis for employing butterfly wings to construct biomimetic nerve grafts and establish Htt lysosome as a crucial regulator for cell-material interactions.

Angiostrongylus Cantonensis-Conditioned Culture Medium Induces Myelin Basic Protein Alterations via Erk1/2 and NF-κB Activation in Rat RSC96 Schwann Cells.

Eating of excessive raw or undercooked environmental snails produces angiostrongyliasis demyelination caused by Angiostrongylus cantonensis (A. cantonensis). The aim of this study was to investigate the association between extracellular signal-regulated kinase (Erk)1/2-nuclear factor (NF)-κB pathway and myelin basic protein (MBP) expression in RSC96 Schwann cells treated with A. cantonensis-conditioned culture medium, which was prepared by culturing the third-stage (L3) nematode larvae in DMEM for 72 h. The supernatants were collected and filtered before use. Our results showed that MBP was produced in the RSC96 cells at 16 h to 48 h post-stimulation (PS). Phosphorylated (p)-NF-κB levels were significantly increased from 8 h to 48 h PS, as were the p-Erk1/2 levels at the same time points. Additionally, expression of p-NF-κB and MBP was significantly decreased by treatment with QNZ, an NF-κB inhibitor. Treatment with PD98059, an Erk kinase inhibitor, efficiently reduced p-Erk1/2, p-NF-κB and MBP expression in the Schwann cells. These results suggest that A. cantonensis-conditioned culture medium induced suppression of the Erk1/2-NF-κB signaling pathway leading to reduced MBP production in RSC96 Schwann cells. Thus, inhibiting this signaling intermediate involved in MBP expression may be a potential method for controlling inflammatory development of A. cantonensis-induced MBP changes in preceded demyelination.

A curcumin derivative J147 ameliorates diabetic peripheral neuropathy in streptozotocin (STZ)-induced DPN rat models through negative regulation AMPK on TRPA1

To investigate the specific molecular mechanisms and effects of curcumin derivative J147 on diabetic peripheral neuropathy (DPN). We constructed streptozotocin (STZ)-induced DPN rat models to detected mechanical withdrawal threshold (MWT) in vivo using Von Frey filaments. In vitro, we measured cell viability and apoptosis, adenosine 5'-monophosphate-activated protein kinase (AMPK) and transient receptor potential A1 (TRPA1) expression using MTT, flow cytometry, qRT-PCR and western blot. Then, TRPA1 expression level and calcium reaction level were assessed in agonist AICAR treated RSC96cells. The results showed that J147reduced MWT in vivo, increased the mRNA and protein level of AMPK, reduced TRPA1 expression and calcium reaction level in AITCR treated RSC96 cells, and had no obvious effect on cell viability and apoptosis. Besides, AMPK negative regulated TRPA1 expression in RSC96 cells. J147 could ameliorate DPN via negative regulation AMPK on TRPA1 in vivo and in vitro. A curcumin derivative J147might be a new therapeutic potential for the treatment of DPN.

Using a Novel Supramolecular Gel Cryopreservation System in Microchannel to Minimize the Cell Injury

The storage of living cells is the major challenge for cell research and cell treatment. Here, we introduced a novel supramolecular gel cryopreservation system which was prepared in the microchannel, and the supramolecular gel (BDTC) was self-assembled by gelator Boc- O-dodecyl-l-tyrosine (BDT). This cryopreservation system could obviously minimize the cell injury because the BDTC supramolecular gel had a more compact three-dimensional network structure when the BDT gelator self-assembled in the confined space of microchannel. This compact structure could confine the growth of the ice crystal, reduce the change rate of cell volumes and osmotic shock, decrease the freezing point of the cryopreservation system, and possess better protection capability. Furthermore, the results of functionality assessments showed that the thawed cells could grow and proliferate well and remain the same growth trend of the fresh cells after the RSC96 cells flowed out from the microchannel. This novel method has potential to be used for the cryopreservation of cells, cell therapy, and tissue engineering.

Hyperglycemia-induced Bcl-2/Bax-mediated apoptosis of Schwann cells via mTORC1/S6K1 inhibition in diabetic peripheral neuropathy

Schwann cell apoptosis is one of the characteristics of diabetic peripheral neuropathy (DPN). The mammalian target of rapamycin (mTOR) is a multifunctional signaling pathway that regulates cell apoptosis in various types of tissues and cells. To investigate whether the mTOR pathway is involved in cell apoptosis in the Schwann cells of DPN, diabetic mice and rat Schwann cells (RSC96) were chosen to detect phospho-mTOR (Ser 2448), phospho-S6K1 (Thr 389), phospho-4EBP1 (Thr 37/46), Bcl-2, Bax and cleaved caspase-3 by diverse pathological and biological techniques. The results showed that phospho-mTOR (Ser 2448) was decreased in the sciatic nerves of diabetic mice, concomitant with decreased Bcl-2, increased Bax, cleaved caspase-3 and cell apoptosis. In addition, high glucose treatment for 72 h caused a 35.95% decrease in the phospho-mTOR (Ser 2448)/mTOR ratio, a 65.50% decrease in the phospho-S6K1 (Thr 389)/S6K1 ratio, a 3.67-fold increase in the Bax/Bcl-2 ratio and a 1.47-fold increase in the cleaved caspase-3/caspase-3 ratio. Furthermore, mTORC1 inhibition, rather than mTORC2 inhibition, resulted in mitochondrial controlled apoptosis in RSC96 cells by silencing RAPTOR or RICTOR. Again, suppression of the mTORC1 pathway by a chemical inhibitor led to mitochondrial controlled apoptosis in cultured RSC96 cells in vitro. By contrast, activation of the mTORC1 pathway with MHY1485 prevented decreased phospho-S6K1 (Thr 389) levels caused by high glucose and cell apoptosis. Additionally, constitutive activation of S6K1 avoided high glucose-induced cell apoptosis in RSC96 cells. In summary, our findings suggest that activating mTORC1/S6K1 signaling in Schwann cells may be a promising strategy for the prevention and treatment of DPN.

Potential biological functions of microvesicles derived from adenoid cystic carcinoma.

Microvesicles (MVs) are secreted by multiple types of tumor cell and are involved in tumor progression and metastasis. The aim of the present study was to explore the effects of MVs derived from salivary adenoid cystic carcinoma (SACC) and to investigate their potential involvement in the pathogenesis of perineural invasion of SACC. MVs were isolated from ACCs cells, and differential gene expression profiles of these MVs were compared with their donor cells to speculate on their biological functions. Several candidate genes were validated using reverse transcription-quantitative polymerase chain reaction analysis. The effects of ACCs MVs on rat Schwann cells (RSC96 cells), which are the principal glia of the peripheral nervous system, were then evaluated by phospho-antibody array performed on RSC96 cells transduced with ACCs MVs. The results indicated that ACCs cells may produce MVs. Microarray-based expression profiles between ACCs cells and their MVs identified 1,355 genes involved in cell adhesion, development and the regulation of apoptosis. In addition, the extracellular signal-regulated protein kinase signal pathway in RSC96 cells may be induced by ACCs-derived MVs. These results may help to elucidate the mechanisms underlying perineural invasion in SACC, and to determine a promising anti-tumor biological therapeutic target.

IRE1\u03b1 siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

Diabetic peripheral neuropathy (DPN) is mainly characterized by demyelination resulted from the apoptosis of the Schwann cell (SCs). Although the exact mechanisms underlying DPN remain unclear, endoplasmic reticulum (ER) stress is strongly implicated in the apoptosis. Under ER stress, activated inositol-requiring kinase 1α (IRE1α) unregulated CHOP, phosphorylated JNK and Caspase-12 to aggravate apoptosis-mediated damage of DPN. Therefore, we tested the hypothesis that inhibition of IRE1α could reduce the ER stress-related apoptosis to relieve DPN. Here, we show that IRE1α siRNA improved the neurological morphology and function of DPN rats and rescued ER stress-related apoptosis in the sciatic nerve. Additionally, RSC96 cells transfected with IRE1α siRNA were used as in vitro model of DPN. It was found that IRE1α siRNA also decreased high glucose-induced apoptosis and inhibited ER stress-related apoptosis in the cells. Altogether, our results suggest that IRE1α should be considered a potential therapeutic agent for DPN.

DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

PurposeImpotence is one of the major complications occurring in prostate cancer patients after radical prostectomy (RP). Self-repair of the injured nerve has been observed in animal models and in patients after RP. However, the downstream signalling is not well documented. Here, we found that the DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation.Materials and MethodsThe 3 groups were a sham group, a 14-day post-bilateral cavernous nerve injury (BCNI) group and a 28-day post-BCNI group. Erectile function was assessed and immunohistochemistry was performed. The rat Schwann cell RSC96 line was chosen for gene knockdown, cell viability, western blot, immunofluorescence and co-immunoprecipitation assays.ResultsThe intracavernosal pressure was low on the 14th day after BCNI and partially increased by the 28th day. GAS6 and p-AXL expression gradually increased in the cavernous nerve after BCNI. RSC96 cells incubated with a GAS6 ligand showed increased levels of p-ERK1/2 and p-AKT. Moreover, DAPK and CIP2A.p-AXL and p-DAPK and CIP2A complexes were identified by both immunoblotting and co-immunoprecipitation.ConclusionThe DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation. CIP2A inhibits PP2A activity, which results in p-DAPK(S308) maintenance and promotes Schwann cell proliferation. CIP2A is a potential target for the treatment of nerve injury after RP.

Gentiopicroside Ameliorates Diabetic Peripheral Neuropathy by Modulating PPAR- Γ/AMPK/ACC Signaling Pathway

Background/Aims: Gentiopicroside is promising as an important secoiridoid compound against pain. The present study aimed to investigate the analgesic effect and the probable mechanism of Gentiopicroside on Diabetic Peripheral Neuropathy (DPN), and to figure out the association among Gentiopicroside, dyslipidemia and PPAR- γ/AMPK/ACC signaling pathway. Methods: DPN rat models were established by streptozotocin and RSC96 cells were cultured. Hot, cold and mechanical tactile allodynia were conducted. Blood lipids, nerve blood flow, Motor Nerve Conduction Velocity (MNCV) and Sensory Nerve Conduction Velocity (SNCV) were detected. Gene and protein expression of PPAR- γ/AMPK/ACC pathway was analyzed by reverse transcription-quan titative polymerase chain reaction (RT-qPCR) and Westernblot. Besides, PPAR-γ antagonist GW9662 and agonist rosiglitazone, AMPK antagonist compound C and activator AICAR as well as ACC inhibitor TOFA were used to further confirm the relationship between PPAR-γ and AMPK. Results: The results demonstrated that Gentiopicroside markedly ameliorated hyperalgesia with prolonged paw withdrawal latency to heat and cold stimuli and fewer responses to mechanical allodynia compared with DPN model group. Gentiopicroside regulated dyslipidemia, enhanced nerve blood flow and improved MNCV as well as SNCV. Gentiopicroside suppressed ACC expression through the activation of AMPK and PPAR-γ mediated the activation of AMPK and subsequent inhibition of ACC expression. Conclusion: In conclusion, the present study demon strated that Gentiopicroside exerted nerve-protective effect and attenuated experimental DPN by restoring dyslipidmia and improved nerve blood flow through regulating PPAR-γ/AMPK/ACC signal pathway. These results provided a promising potential treatment of DPN.