Electroacupuncture improves peripheral neuropathy by up-regulating Sirt1/PGC-1α/TFAM pathway in type 2 diabetes rats with peripheral neuropathy. / 基于Sirt1/PGC-1α/TFAM通路探讨电针治疗2åž‹ç³–å°¿ç— å‘¨å›´ç¥žç»ç— å˜çš„æœºåˆ¶.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on activation of silent information regulator 1 (Sirt1)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)/mitochondrial transcription factor A (TFAM) pathway in type 2 diabetes (T2DM) rats with peripheral neuropathy (DPN) , so as to explore its possible mechanisms underlying improvement of DPN. METHODS: Thirty male SD rats were randomly divided into blank control group (n=8) and DPN model group (n=22) which were further divided into model group (n=8) and EA group (n=8) after successful modeling. The model of T2DM was established by high-fat diet and low-dose intraperitoneal injection of streptozocin (35 mg/kg). For rats of the EA group (anesthetized with isoflurane), EA stimulation (2 Hz/15 Hz, 2 mA) was applied to "Tianshu"(ST25) for 20 min, once daily, 6 times a week for 6 weeks. The blood glucose level, body weight, area under curve (AUC) of glucose tolerance test, and hind-paw mechanical pain threshold and thermal pain threshold were observed. The intra-epidermal nerve fiber density (IENFD) of the hind-foot pad was observed by immunofluorescence staining. The motor nerve conduction velocity (MNCV) of the sciatic nerve was measured by using electrophysiological method. H.E. staining was used to observe the histopathological changes of the sciatic nerve after modeling. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of the sciatic nerve. The protein expressions of energy-related Sirt1, PGC-1α and TFAM in the sciatic nerve was detected by Western blot. RESULTS: Compared with the blank control group, the model group had a higher blood glucose contents and AUC (P<0.001), a slower MNCV (P<0.01), and a decrease in the body weight and in the mechanical and thermal pain thresholds (P<0.001) and IENFD (P<0.001), and in the expression levels of Sirt1, PGC-1α and TFAM (P<0.05, P<0.01). In contrast to the model group, the EA group had a decrease in the blood glucose contents and AUC (P<0.05, P<0.01), and an increase in mechanical and thermal pain thresholds, MNCV, IENFD, and expression levels of Sirt1, PGC-1α and TFAM proteins (P<0.01, P<0.05). In addition, results of histopathological and ultrastructural changes of the sciatic nerve showed more fragmented and disordered distribution of axons on the transverse section, and extensive separation of myelin and axons, uneven myelin thickness, axonal degeneration and irregular shape in the model group, whereas in the EA group, the axons on the transverse section were relatively more dense and more complete, the myelin sheath of the sciatic nerve was relatively uniform, and the axonal shape was relatively regular with relatively milder lesions. CONCLUSIONS: EA up-regulates the expressions of Sirt1, PGC-1α, TFAM in T2DM rats with DPN, which may be associated with its functions in improving and repairing the injured peripheral nerves in rats with DPN.

NTPDase1-ATP-P2Y2Rs axis in the sciatic nerve contributes to acupuncture at "Zusanli" (ST36)-induced analgesia in ankle arthritis rats.

BACKGROUND: The efficacy of acupuncture at Zusanli (ST36) in alleviating lower-limb pain is widely acknowledged in clinical practice, while its underlying mechanism remains incompletely elucidated. Our previous research had revealed that the prompt analgesia induced by needling-ST36 was accompanied by expression alterations in certain exco-nucleotidases within the sciatic nerve. Building upon this finding, the current work focused on NTPDase1, the primary ecto-nucleotidase in the human body, which converts ATP into AMP. METHODS: A 20-min acupuncture was administered unilaterally at the ST36 on rats with acute ankle arthritis. The pain thresholds of the injured hind paws were determined. Pharmacological interference was carried out by introducing the corresponding reagents to the sciatic nerve. ATP levels around the excised nerve were measured using a luciferase-luciferin assay. Live calcium imaging, utilizing the Fura 2-related-F340/F380 ratio, was conducted on Schwann cells in excised nerves and cultured rat SCs line, RSC96 cells. RESULTS: The analgesic effect induced by needling-ST36 was impaired when preventing ATP degradation via inhibiting NTPDase1 activities with ARL67156 or Ticlopidine. Conversely, increasing NTPDase1 activities with Apyrase duplicated the acupuncture effect. Similarly, preventing the conversion of AMP to adenosine via suppression of NT5E with AMP-CP hindered the acupuncture effect. Unexpectedly, impeded ATP hydrolysis ability and diminished NTPDase1 expression were observed in the treated group. Agonism at P2Y2Rs with ATP, UTP, or INS365 resulted in anti-nociception. Contrarily, antagonism at P2Y2Rs with Suramin or AR-C 118925xx prevented acupuncture analgesia. Immunofluorescent labeling demonstrated that the treated rats expressed more P2Y2Rs that were predominant in Schwann cells. Suppression of Schwann cells by inhibiting ErbB receptors also prevented acupuncture analgesia. Finally, living imaging on the excised nerves or RSC96 cells showed that agonism at P2Y2Rs indeed led to [Ca2+]i rise. CONCLUSION: These findings strongly suggest that the analgesic mechanism of needling-ST36 on the hypersensation in the lower limb partially relies on NTPDase1 activities in the sciatic nerve. In addition to facilitating adenosine signaling in conjunction with NT5E, most importantly, NTPDase1 may provide an appropriate low-level ATP milieu for the activation of P2Y2R in the sciatic nerve, particularly in Schwann cells.

Electroacupuncture Promotes Nerve Regeneration and Functional Recovery Through Regulating lncRNA GAS5 Targeting miR-21 After Sciatic Nerve Injury.

Although the benefits of electroacupuncture (EA) for peripheral nerve injury (PNI) are well accepted in clinical practice, the underlying mechanism remains incompletely elucidated. In our study, we observed that EA intervention led to a reduction in the expression of the long non-coding RNA growth-arrest-specific transcript 5 (GAS5) and an increased in miR-21 levels within the injured nerve, effectively promoting functional recovery and nerve regeneration following sciatic nerve injury (SNI). In contrast, administration of adeno-associated virus expressing GAS5 (AAV-GAS5) weakened the therapeutic effect of EA. On the other hand, both silencing GAS5 and introducing a miR-21 mimic prominently enhanced the proliferation activity and migration ability of Schwann cells (SCs), while also inhibiting SCs apoptosis. On the contrary, inhibition of SCs apoptosis was found to be mediated by miR-21. Additionally, overexpression of GAS5 counteracted the effects of the miR-21 mimic on SCs. Moreover, SCs that transfected with the miR-21 mimic promoted neurite growth in hypoxia/reoxygenation-induced neurons, which might be prevented by overexpressing GAS5. Furthermore, GAS5 was found to be widely distributed in the cytoplasm and was negatively regulated by miR-21. Consequently, the targeting of GAS5 by miR-21 represents a potential mechanism through which EA enhances reinnervation and functional restoration following SNI. Mechanistically, the GAS5/miR-21 axis can modulate the proliferation, migration, and apoptosis of SCs while potentially influencing the neurite growth of neurons.

RTA-408 Regulates p-NF-κB/TSLP/STAT5 Signaling to Ameliorate Nociceptive Hypersensitivity in Chronic Constriction Injury Rats.

Neuropathic pain following nerve injury is a complex condition, which often puts a negative impact on life and remains a sustained problem. To make pain management better is of great significance and unmet need. RTA 408 (Omaveloxone) is a traditional Asian medicine with a valid anti-inflammatory property. Thus, we aim to investigate the therapeutic effect of RTA-408 on mechanical allodynia in chronic constriction injury (CCI) rats as well as the underlying mechanisms. Neuropathic pain was induced by using CCI of the rats' sciatic nerve (SN) and the behavior testing was measured by calibrated forceps testing. Activation of Nrf-2, the phosphorylation of nuclear factor-κB (NF-κB), and the inflammatory response were assessed by western blots. The number of apoptotic neurons and degree of glial cell reaction were examined by immunofluorescence assay. RTA-408 exerts an analgesic effect on CCI rats. RTA-408 reduces neuronal apoptosis and glial cell activation by increasing Nrf-2 expression and decreasing the inflammatory response (TNF-α/ p-NF-κB/ TSLP/ STAT5). These data suggest that RTA-408 is a candidate with potential to reduce nociceptive hypersensitivity after CCI by targeting TSLP/STAT5 signaling.

Acetyl-11-Keto-Beta-Boswellic Acid Activates the Nrf2/HO-1 Signaling Pathway in Schwann Cells to Reduce Oxidative Stress and Promote Sciatic Nerve Injury Repair.

Boswellia is a traditional medicine for bruises and injuries. Its main active ingredient, acetyl-11-keto-beta-boswellic acid, has antioxidant and antiapoptotic effects. In this experiment, we used Sprague-Dawley rats to make a sciatic nerve injury model to detect the transcription factor NF-E2-related factor 2/heme oxygenase 1 signaling pathway and apoptosis, combined with clinical indicators, for testing whether acetyl-11-keto-beta-boswellic acid can reduce oxidative stress and promote sciatic nerve repair. Our results showed that acetyl-11-keto-beta-boswellic acid administration promoted myelin regeneration and functional recovery in the rat sciatic nerve, reduced lipid peroxidation levels, upregulated the expression of various antioxidant enzymes and enhanced enzyme activity, decreased the expression levels of apoptosis-related proteins, and promoted nuclear translocation of the transcription factor NF-E2-related factor 2 protein. In vitro studies revealed that acetyl-11-keto-beta-boswellic acid reduced H2O2-induced reactive oxygen species production, restored mitochondrial membrane potential, upregulated the expression of various antioxidant enzymes, and downregulated apoptosis-related indicators in Schwann cells, and these therapeutic effects of acetyl-11-keto-beta-boswellic acid were reversed after ML385 treatment in Schwann cells. In summary, acetyl-11-keto-beta-boswellic acid alleviates oxidative stress and apoptosis caused by sciatic nerve injury in rats by activating the transcription factor NF-E2-related factor 2/heme oxygenase 1 signaling pathway, promotes the recovery of sciatic nerve function in rats, and is a promising therapeutic agent to promote sciatic nerve repair by alleviating excessive oxidative stress.

Glycolytic System in Axons Supplement Decreased ATP Levels after Axotomy of the Peripheral Nerve.

Wallerian degeneration (WD) occurs in the early stages of numerous neurologic disorders, and clarifying WD pathology is crucial for the advancement of neurologic therapies. ATP is acknowledged as one of the key pathologic substances in WD. The ATP-related pathologic pathways that regulate WD have been defined. The elevation of ATP levels in axon contributes to delay WD and protects axons. However, ATP is necessary for the active processes to proceed WD, given that WD is stringently managed by auto-destruction programs. But little is known about the bioenergetics during WD. In this study, we made sciatic nerve transection models for GO-ATeam2 knock-in rats and mice. We presented the spatiotemporal ATP distribution in the injured axons with in vivo ATP imaging systems, and investigated the metabolic source of ATP in the distal nerve stump. A gradual decrease in ATP levels was observed before the progression of WD. In addition, the glycolytic system and monocarboxylate transporters (MCTs) were activated in Schwann cells following axotomy. Interestingly, in axons, we found the activation of glycolytic system and the inactivation of the tricarboxylic acid (TCA) cycle. Glycolytic inhibitors, 2-deoxyglucose (2-DG) and MCT inhibitors, a-cyano-4-hydroxycinnamic acid (4-CIN) decreased ATP and enhanced WD progression, whereas mitochondrial pyruvate carrier (MPC) inhibitors (MSDC-0160) did not change. Finally, ethyl pyruvate (EP) increased ATP levels and delayed WD. Together, our findings suggest that glycolytic system, both in Schwann cells and axons, is the main source of maintaining ATP levels in the distal nerve stump.

Antinociceptive effect of ethanolic extract of Bauhinia brachycarpa Benth on neuropathic pain model induced by partial sciatic nerve ligation.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Bauhinia brachycarpa Benth (BBB) has been traditionally used for treating muscle aches such as bone pain, and neuralgia for a long time, on account of its sedative and antinociceptive activities in Yunnan province of China. However, there was no experimental evidence to confirm its traditional medicinal use. AIM OF THE STUDY: The antinociceptive effect and possible mechanism of ethanolic extract of BBB on neuropathic pain was evaluated through a model of partial sciatic nerve ligation in mice. MATERIALS AND METHODS: A commonly employed animal model induced by partial sciatic nerve ligation (PSNL) in mice was established in the aim of studying neuropathic pain. Ethanolic extract of BBB (1000, 500, 250 mg/kg) and pregabalin (60 mg/kg) were intragastric administrated daily for 7 days post-PSNL. Mechanical and thermal hyperalgesia were assessed throughout the experimental period. After the experiment, the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in serum were determined by ELISA. The mRNA expressions of ionized calcium binding adaptor molecule 1 (Iba-1), CD16, CD206, arginine-1 (Arg-1), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS) in the spinal cord were detected by qPCR. The protein levels of Iba-1, CD16, CD206, and p38 phosphorylation in the spinal cord were detected by immunohistochemistry and western blotting. The phytochemical analysis of BBB was performed through the colorimetric test. RESULTS: Neuropathic pain induced by PSNL was significantly alleviated by BBB treatment, which decreased pro-inflammatory cytokines such as TNF-α, IL-1ß and iNOS, increased anti-inflammatory cytokine such as IL-10 and Arg-1, and attenuated p38 phosphorylation. BBB also reduced the number of Iba-1 and CD16 positive cells, but it enhanced the number of CD206 positive cells. n-Butanol portion that was partitioned from the ethanolic extract had the highest content of total flavonoids among all the portions, and the antinociceptive effect of n-butanol portion is better than that of other portions. CONCLUSIONS: Our findings indicate that the antinociceptive effect of BBB is mediated by inhibiting the inflammatory response and regulating the differentiation of microglia. The antinociceptive effect of BBB was related to the content of total flavonoids.

Based on proteomics to explore the mechanism of mecobalamin promoting the repair of injured peripheral nerves.

Mecobalamin is commonly used in the adjuvant intervention of various peripheral nerve injuries. Actin cytoskeleton plays a role in the regeneration of myelin and axon. Therefore, the purpose of this study was to explore the possibility of mecobalamin regulating actin cytoskeleton in repairing nerve injury. In this study, a crush injury on the right sciatic nerve of two groups of rats (12 in each group) was established. The control group was only given normal saline (i.g.), and the intervention group was given mecobalamin 1 mg/kg (i.g.). The rats were sacrificed on 28th day and the injured nerves were collected for proteomics. The result shows that regulation of actin cytoskeleton pathway changed significantly. The expression of protein Vav1 was verified by Western blot and immunofluorescence. In the intervention group, the nerve fiber structure was complete, the axons were dense and symmetrical, and the myelin sheath was compact and uniform in thickness. The positive rate of myelin basic protein and ßⅢ-tubulin was higher than that in the control group. The findings of the study show that mecobalamin regulates the actin cytoskeleton in the repair of nerve damage and upregulates Vav1 in the regulation of actin cytoskeleton pathway.

Local vibration therapy promotes the recovery of nerve function in rats with sciatic nerve injury.

OBJECTIVE: It has been reported that local vibration therapy can benefit recovery after peripheral nerve injury, but the optimized parameters and effective mechanism were unclear. In the present study, we investigated the effect of local vibration therapy of different amplitudes on the recovery of nerve function in rats with sciatic nerve injury (SNI). METHODS: Adult male Sprague-Dawley rats were subjected to SNI and then randomly divided into 5 groups: sham group, SNI group, SNI + A-1 mm group, SNI + A-2 mm group, and SNI + A-4 mm group (A refers to the amplitude; n = 10 per group). Starting on the 7th day after model initiation, local vibration therapy was given for 21 consecutive days with a frequency of 10 Hz and an amplitude of 1, 2 or 4 mm for 5 min. The sciatic function index (SFI) was assessed before surgery and on the 7th, 14th, 21st and 28th days after surgery. Tissues were harvested on the 28th day after surgery for morphological, immunofluorescence and Western blot analysis. RESULTS: Compared with the SNI group, on the 28th day after surgery, the SFIs of the treatment groups were increased; the difference in the SNI + A-2 mm group was the most obvious (95% confidence interval [CI]: [5.86, 27.09], P < 0.001), and the cross-sectional areas of myocytes in all of the treatment groups were improved. The G-ratios in the SNI + A-1 mm group and SNI + A-2 mm group were reduced significantly (95% CI: [-0.12, -0.02], P = 0.007; 95% CI: [-0.15, -0.06], P < 0.001). In addition, the expressions of S100 and nerve growth factor proteins in the treatment groups were increased; the phosphorylation expressions of ERK1/2 protein in the SNI + A-2 mm group and SNI + A-4 mm group were upregulated (95% CI: [0.03, 0.96], P = 0.038; 95% CI: [0.01, 0.94], P = 0.047, respectively), and the phosphorylation expression of Akt in the SNI + A-1 mm group was upregulated (95% CI: [0.11, 2.07], P = 0.031). CONCLUSION: Local vibration therapy, especially with medium amplitude, was able to promote the recovery of nerve function in rats with SNI; this result was linked to the proliferation of Schwann cells and the activation of the ERK1/2 and Akt signaling pathways.

Divanillyl sulfone suppresses NLRP3 inflammasome activation via inducing mitophagy to ameliorate chronic neuropathic pain in mice.

BACKGROUND: Chronic neuropathic pain is a frequent sequel to peripheral nerve injury and maladaptive nervous system function. Divanillyl sulfone (DS), a novel structural derivative of 4,4'-dihydroxydibenzyl sulfoxide from a traditional Chinese medicine Gastrodia elata with anti-nociceptive effects, significantly alleviated neuropathic pain following intrathecal injection. Here, we aimed to investigate the underlying mechanisms of DS against neuropathic pain. METHODS: A chronic constrictive injury (CCI) mouse model of neuropathic pain induced by sciatic nerve ligation was performed to evaluate the effect of DS by measuring the limb withdrawal using Von Frey filament test. Immunofluorescence staining was used to assess the cell localizations and expressions of Iba-1, ASC, NLRP3, and ROS, the formation of autolysosome. The levels of NLRP3-related proteins (caspase-1, NLRP3, and IL-1ß), mitophagy-related proteins (LC3, Beclin-1, and p62), and apoptosis-related proteins (Bcl-XL and Bax) were detected by Western blotting. The apoptosis of BV-2 cell and caspase activity were evaluated by flow cytometry. RESULTS: DS significantly alleviated the neuropathic pain by increasing the mechanical withdrawal threshold and inhibiting the activation of NLRP3 in CCI-induced model mice. Our findings indicated that DS promoted the mitophagy by increasing the LC3II and Beclin 1 and decreasing the levels of p62 protein in BV-2 cell. This is accompanied by the inhibition of NLRP3 activation, which was shown as inhibited the expression of NLRP3 in lysates as well as the secretion of mature caspase-1 p10 and IL-1ß p17 in supernatants in cultured BV-2 microglia. In addition, DS could promote mitophagy-induced improvement of dysfunctional mitochondria by clearing intracellular ROS and restoring mitochondrial membrane potential. CONCLUSION: Together, our findings demonstrated that DS ameliorate chronic neuropathic pain in mice by suppressing NLRP3 inflammasome activation induced by mitophagy in microglia. DS may be a promising therapeutic agent for chronic neuropathic pain.

Bixin attenuates mechanical allodynia, anxious and depressive-like behaviors associated with experimental diabetes counteracting oxidative stress and glycated hemoglobin.

Neuropathic pain, depression, and anxiety are common comorbidities in diabetic patients, whose pathophysiology involves hyperglycemia-induced increased oxidative stress. Bixin (BIX), an apocarotenoid extracted from the seeds of Bixa orellana, has been used in traditional medicine to treat diabetes and has been recognized by its antioxidant profile. We aimed to investigate the effect of the BIX over the mechanical allodynia, depressive, and anxious-like behaviors associated with experimental diabetes, along with its involved mechanisms. Streptozotocin-induced diabetic rats were treated for 17 days (starting 14 days after diabetes induction) with the corresponding vehicle, BIX (10, 30 or 90 mg/kg; p.o), or INS (6 IU; s.c.). Mechanical allodynia, depressive, and anxious-like behavior were assessed by electronic Von Frey, forced swimming, and elevated plus-maze tests, respectively. Locomotor activity was assessed by the open field test. Blood glycated hemoglobin (HbA1) and the levels of lipid peroxidation (LPO) and reduced glutathione (GSH) were evaluated on the hippocampus, pre-frontal cortex, lumbar spinal cord, and sciatic nerve. Diabetic animals developed mechanical allodynia, depressive and anxious-like behavior, increased plasma HbA1, increased LPO, and decreased GSH levels in tissues analyzed. Repeated BIX-treatment (at all tested doses) significantly attenuated mechanical allodynia, the depressive (30 and 90 mg/kg) and, anxious-like behaviors (all doses) in diabetic rats, without changing the locomotor performance. BIX (at all tested doses) restored the oxidative parameters in tissues analyzed and reduced the plasma HbA1. Thereby, bixin may represent an alternative for the treatment of comorbidities associated with diabetes, counteracting oxidative stress and plasma HbA1.

Bromelain reduced pro-inflammatory mediators as a common pathway that mediate antinociceptive and anti-anxiety effects in sciatic nerve ligated Wistar rats.

The involvement of pro-inflammatory mediators complicates the complex mechanism in neuropathic pain (NP). This study investigated the roles of bromelain against pro-inflammatory mediators as a mechanism that underpins its antinociceptive and anti-anxiety effects in the peripheral model of NP. Sixty-four male Wistar rats randomly divided into eight groups, were used for the study. A chronic constriction injury model of peripheral neuropathy was used to induce NP. Tail-immersion and von Frey filaments tests were used to assess hyperalgesia while open field and elevated plus mazes were used to assess anxiety-like behaviour. NF-кB, iNOS, nitrate, and pro-inflammatory cytokines were investigated in the plasma, sciatic nerve, and brain tissues using ELISA, spectrophotometer, and immunohistochemistry techniques after twenty-one days of treatment. Bromelain significantly (p < 0.05) improved the cardinal signs of NP and inhibited anxiety-like behaviours in ligated Wistar rats. It mitigated the increases in cerebral cortex interleukin (IL) -1ß, IL-6, and PGE2 levels. Bromelain reduced NF-кB, IL-1ß, IL-6, TNF-α, PGE2, and nitrate concentrations as well as the expression of iNOS in the sciatic nerve. Hence, the antinociceptive and anxiolytic effects of bromelain in the sciatic nerve ligation model of NP is in part due to its ability to reduce nitrosative and inflammatory activities.

Jinmaitong ameliorates diabetes-induced peripheral neuropathy in rats through Wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinmaitong (JMT) is a prescription of Traditional Chinese Medicine, which is composed of ten herbal drugs and two animal drugs. It has long been used for the treatment of diabetic peripheral neuropathy (DPN). AIM OF STUDY: Wnt/ß-catenin pathway is considered as an essential and direct driver of myelinogenesis. This study aims to evaluate the protective effect of JMT against DPN dynamically during a 16-weeks' treatment, and to investigate the underlying mechanism in which the Wnt/ß-catenin pathway is involved. MATERIALS AND METHODS: Diabetic model was induced by single intraperitoneal injection of Streptozotocin (STZ) using male Sprague-Dawley rats. The model rats were divided into five groups and administrated with JMT at three doses (0.437, 0.875, and 1.75 g/kg per day), neurotropin (positive drug, 2.67 NU/kg per day), and placebo (deionized water), respectively, for continuous 8 weeks (n = 9-10), 12 weeks (n = 8-10), or 16 weeks (n = 7-9). Meanwhile, rats in control group were administrated with placebo (n = 10 for 8 weeks, n = 9 for 12 and 16 weeks, respectively). Blood glucose and body weight were monitored every four weeks. Mechanical allodynia was assessed using mechanical withdrawal threshold (MWT) test. The morphological change of sciatic nerves were observed by transmission electron microscope (TEM) and hematoxylin and eosin (HE) stain. The mRNA and protein levels of targeted genes were evaluated by quantitative real time-PCR and western bolt, respectively. Myelin protein zero (MPZ) and mediators involved in Wnt/ß-catenin pathway, such as ß-catenin, glycogen synthase kinase 3ß (GSK-3ß), and WNT inhibitory factor-1 (WIF-1), were compared among different groups after treatment of 8, 12, and 16 weeks, respectively. RESULTS: The mechanical allodynia and peripheral nerve morphology were degenerated in DPN rats over time, and notably improved after JMT-treatment of 12 and 16 weeks. The decreased MPZ level in DPN rats were also significantly amended by JMT. More importantly, we found that the suppressed Wnt/ß-catenin pathway in sciatic nerves of DPN rats was overtly up-regulated by JMT in a time-dependent manner. Among the three doses, JMT at the middle dose showed the best effect. CONCLUSIONS: JMT effectively ameliorated diabetic-induced peripheral neuropathy, which was mediated by the activation of Wnt/ß-catenin signaling pathway. This study provided new perspective to understand the neuroprotective mechanism of JMT.

Oral administration of Cystine and Theanine ameliorates oxaliplatin-induced chronic peripheral neuropathy in rodents.

Oxaliplatin frequently causes severe peripheral neuropathy as a dose-limiting toxicity. However, this toxicity lacks a strategy for prevention. Cystine/Theanine is a supplement, which includes precursors for the biosynthesis of glutathione. In this study, we investigated the effects of Cystine/Theanine on oxaliplatin-induced peripheral neuropathy using an in vivo model. Repeated injection of oxaliplatin (4 mg/kg intraperitoneally twice a week for 2 weeks) caused mechanical allodynia, cold hyperalgesia and axonal degeneration of the sciatic nerve in rats. Mechanical allodynia and axonal degeneration, but not cold hyperalgesia, were ameliorated by daily co-administration of Cystine [200 mg/kg orally (p.o.)] and Theanine (80 mg/kg p.o.). Moreover, co-administration of Cystine and Theanine to rats significantly increased the glutathione level in the sciatic nerve compared with the oxaliplatin group. Furthermore, Cystine and Theanine did not attenuate the tumour cytotoxicity of oxaliplatin in C-26 tumour cell-bearing mice. These findings suggest that Cystine and Theanine may be beneficial for preventing oxaliplatin-induced peripheral neuropathy.

Augmented Buyang Huanwu Decoction facilitates axonal regeneration after peripheral nerve transection through the regulation of inflammatory cytokine production.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal formulation Buyang Huanwu Decoction (BYHWD) has been used to treat cardiovascular disorders including cerebral ischemia. Recent studies showed its effects on promoting axonal regeneration after nerve injury. However, compositional reformulation supplemented with herbal components that regulates inflammation may increase its efficacy for nerve repair. AIM OF THE STUDY: We prepared a new herbal decoction by adding selected herbal components to BYHWD (augmented BYHWD; ABHD) and investigated the effect of ABHD on the production of inflammatory cytokines and axonal regeneration using an animal model of nerve transection and coaptation (NTC). MATERIALS AND METHODS: A rat model of NTC was performed on the sciatic nerve. The sciatic nerve and dorsal root ganglion (DRG) were isolated and used for immunofluorescence staining and western blot analysis. DRG tissue was also used to prepare primary neuron culture and the length of neurites was analyzed. Sensorimotor nerve activities were assessed by rotarod and von Frey tests. RESULTS: Three herbal components that facilitated neurite outgrowth were chosen to formulate ABHD. ABHD administration into the sciatic nerve 1 week or 3 months after NTC facilitated axonal regeneration. Cell division cycle 2 (Cdc2) and brain-derived neurotrophic factor (BDNF) proteins were induced from the reconnected distal portion of the sciatic nerve and the levels were further elevated by in vivo administration of ABHD. Phospho-Erk1/2 level was increased by ABHD treatment as well, implying its role in mediating retrograde transport of BDNF signals into the neuronal cell body. Production of inflammatory cytokines IL-1ß and TNF-α was induced in the reconnected nerve but attenuated by ABHD treatment. Behavioral tests revealed that ABHD treatment improved functional recovery of sensorimotor activities. CONCLUSIONS: A newly formulated ABHD is effective at regulating the production of inflammatory cytokines and promoting axonal regeneration after nerve transection and may be considered to develop therapeutic strategies for peripheral nerve injury disorders.

Loganin prevents chronic constriction injury-provoked neuropathic pain by reducing TNF-α/IL-1ß-mediated NF-κB activation and Schwann cell demyelination.

BACKGROUND: Peripheral nerve injury can produce chronic and ultimately neuropathic pain. The chronic constriction injury (CCI) model has provided a deeper understanding of nociception and chronic pain. Loganin is a well-known herbal medicine with glucose-lowering action and neuroprotective activity. PURPOSE: This study investigated the molecular mechanisms by which loganin reduced CCI-induced neuropathic pain. METHODS: Sprague-Dawley rats were randomly divided into four groups: sham, sham+loganin, CCI and CCI+loganin. Loganin (1 or 5 mg/kg/day) was injected intraperitoneally once daily for 14 days, starting the day after CCI. For behavioral testing, mechanical and thermal responses were assessed before surgery and on d1, d3, d7 and d14 after surgery. Sciatic nerves (SNs) were collected to measure proinflammatory cytokines. Proximal and distal SNs were collected separately for Western blotting and immunofluorescence studies. RESULTS: Thermal hyperalgesia and mechanical allodynia were reduced in the loganin-treated group as compared to the CCI group. Loganin (5 mg/kg/day) prevented CCI from inducing proinflammatory cytokines (TNF-α, IL-1ß), inflammatory proteins (TNF-α, IL-1ß, pNFκB, pIκB/IκB, iNOS) and receptor (TNFR1, IL-1R), adaptor protein (TRAF2) of TNF-α, and Schwann cell demyelination and axonal damage. Loganin also blocked IκB phosphorylation (p-IκB). Double immunofluorescent staining further demonstrated that pNFκB/pIκB protein was reduced by loganin in Schwann cells on d7 after CCI. In the distal stumps of injured SN, Schwann cell demyelination was correlated with pain behaviors in CCI rats. CONCLUSION: Our findings indicate that loganin improves CCI-induced neuroinflammation and pain behavior by downregulating TNF-α/IL-1ß-dependent NF-κB activation.

Neuroprotective Effects of Ozone Therapy After Sciatic Nerve Cut Injury.

A number of antioxidants have been used to treat peripheral nerve injury. However, there are few definitive experimental studies of ozone therapy for peripheral nerve cut injury. We aimed to examine the effects of mild level ozone therapy on sciatic nerve regeneration. One hundred adult male Wistar albino rats were randomly divided into four groups: group 1 (n=20) no cut injury or therapy; group 2 (n=20) sham; group 3 (n=30) nerve cut injury, no therapy; group 4 (n=30) nerve cut injury and ozone therapy. Sciatic functional index (SFI) and withdrawal reflex (WDR) were measured for all groups before nerve cut, at postoperative day 1, and at weeks 2, 4, 6 and 8. More myelinated (M) nerve fibers were observed after nerve cut injury in the ozone-therapy group. Significant differences were seen in plasma SOD (superoxide dismutase), CAT (catalase) and GPx (glutathione peroxidase) activities (p<0.05), and significant functional improvement was observed at postoperative weeks 2 and 4 (p<0.05) after ozone treatment. This is the first study conducted for the purpose of examining the effects of ozone therapy on sciatic nerve cut injury.

Prolonged Duration Local Anesthesia by Combined Delivery of Capsaicin- and Tetrodotoxin-Loaded Liposomes.

BACKGROUND: Capsaicin, the active component of chili peppers, can produce sensory-selective peripheral nerve blockade. Coadministration of capsaicin and tetrodotoxin, a site-1 sodium channel blocker, can achieve a synergistic effect on duration of nerve blocks. However, capsaicin can be neurotoxic, and tetrodotoxin can cause systemic toxicity. We evaluated whether codelivery of capsaicin and tetrodotoxin liposomes can achieve prolonged local anesthesia without local or systemic toxicity. METHODS: Capsaicin- and tetrodotoxin-loaded liposomes were developed. Male Sprague-Dawley rats were injected at the sciatic nerve with free capsaicin, capsaicin liposomes, free tetrodotoxin, tetrodotoxin liposomes, and blank liposomes, singly or in combination. Sensory and motor nerve blocks were assessed by a modified hotplate test and a weight-bearing test, respectively. Local toxicity was assessed by histologic scoring of tissues at the injection sites and transmission electron microscopic examination of the sciatic nerves. Systemic toxicity was assessed by rates of contralateral nerve deficits and/or mortality. RESULTS: The combination of capsaicin liposomes and tetrodotoxin liposomes achieved a mean duration of sensory block of 18.2 hours (3.8 hours) [mean (SD)], far longer than that from capsaicin liposomes [0.4 hours (0.5 hours)] (P < .001) or tetrodotoxin liposomes [0.4 hours (0.7 hours)] (P < .001) given separately with or without the second drug in free solution. This combination caused minimal myotoxicity and muscle inflammation, and there were no changes in the percentage or diameter of unmyelinated axons. There was no systemic toxicity. CONCLUSIONS: The combination of encapsulated tetrodotoxin and capsaicin achieved marked prolongation of nerve block. This combination did not cause detectable local or systemic toxicity. Capsaicin may be useful for its synergistic effects on other formulations even when used in very small, safe quantities.

Therapeutic effects of moxibustion simultaneously targeting Nrf2 and NF-κB in diabetic peripheral neuropathy.

Moxibustion is the main alternative medicine treatment that has been beneficial to diabetic peripheral neuropathy (DPN), a common complication secondary to diabetic microvascular injury. However, the underlying protective mechanism of moxibustion against neuroinflammation remains unclear. We hypothesized that moxibustion treats DPN by regulating the balance of nuclear factor-2 erythroid-related factor-2 (Nrf2)-nuclear factor-kappa light chain enhancer of B cells (NF-кB). In vivo, diabetes was induced in rats by injecting streptozotocin (STZ; 60 mg/kg; i.p.). Moxibustion was then applied to "Zusanli" (ST 36), "Guanyuan" (BL 26), and "Yishu" (EX-B 3) acupuncture points. Nerve conduction was detected. Serum interleukin (IL)-1ß, IL-6, and IL-8 levels were determined through enzyme-linked immunosorbent assay. NF-κB and Nrf2 proteins were examined through immunoblot analysis. The mRNA of NF-κB and Nrf2 was evaluated through RT-PCR. We found that the conduction velocity and amplitude of the action potentials of sciatic nerve conduction were reduced in the DPN model group but were rescued by moxibustion treatment. Moxibustion also improved the effect of DPN on other parameters, including ultrastructural changes, NF-κB and Nrf2 expression in the sciatic nerve, and serum IL-1ß, IL-6, and IL-8 levels. Our data suggested that moxibustion may alleviate neuroinflammation by inhibiting NF-κB and by activating Nrf2. Moxibustion may also provide therapeutic effects for patients with DPN by simultaneously targeting Nrf2 and NF-κB.

Wrapping With Basic Fibroblast Growth Factor-Impregnated Collagen Sheet Reduces Rat Sciatic Nerve Allodynia.

Autologous vein wrapping is used to treat recurrent chronic constriction neuropathy and traumatic peripheral nerve injury. However, its use is restricted due to the inability to obtain sufficiently long veins for larger grafts. We previously reported that vein-derived basic fibroblast growth factor (bFGF) promotes heme oxygenase-1 (HO-1), which reduces allodynia via its anti-inflammatory properties. To mimic vein wrapping, we developed a collagen sheet impregnated with bFGF. Chronic constriction injury (CCI) was induced in male Wistar rats as a model of sciatic nerve injury, and the rats were divided into three groups: (i) untreated after CCI surgery (control group), (ii) treated with a collagen sheet wrap impregnated with phosphate-buffered saline (PBS/CS group), and (iii) treated with a collagen sheet wrap impregnated with bFGF (bFGF/CS group). Pain behavior (von Frey test) was evaluated on postoperative days (PODs) 1, 5, 7, and 14. Quantitative polymerase chain reaction was conducted on sciatic nerve RNA to quantify HO-1 gene, Hmox1, expression. Enzyme-linked immunosorbent assay were used to determine HO-1 protein levels on POD 1. von Frey testing showed significantly greater pain hypersensitivity in the control and PBS/CS groups than the bFGF/CS group. In the bFGF/CS group, Hmox1 messenger RNA and HO-1 protein levels were significantly increased in the sciatic nerve compared with the control and PBS/CS groups on PODs 1 and 5 and POD 1, respectively. The bFGF/CS group showed decreased allodynia and HO-1 induction, as observed with vein wrapping. Therefore, local application of bFGF may be an alternative treatment strategy for compressive neuropathy and peripheral nerve trauma in clinical settings. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2258-2263, 2019.