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Background: Renal tubular injury is the main feature of diabetic nephropathy (DN). We intend to investigate the function and related mechanisms of lncRNA SOX2 overlapping transcript (SOX2OT) in high glucose (HG)‐induced oxidative stress and apoptosis of renal tubular epithelial cells (RTECs). Methods: To construct diabetes models, the human kidney‐2 (HK‐2) cells were treated with HG (30 mM), and mice were injected with streptozotocin. The levels of intracellular and mitochondrial reactive oxygen species (ROS) were assessed by dihydroethidium staining and MitoSox staining. The cell apoptosis was assessed by flow cytometry and TUNEL staining. Levels of serum creatinine, blood urea nitrogen (BUN), Urinary ACR, and oxidative stress marker 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) were detected by relevant kits. In addition, fluorescence in situ hybridization staining, RNA‐pull down, RNA immunoprecipitation (RIP), co‐immunoprecipitation (co‐IP), dual‐luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) were also executed. Results: Levels of SOX2OT and silent information regulator 1 (SIRT1) were down‐regulated in HG‐cultured HK‐2 cells. Overexpressing SOX2OT reduced intracellular and mitochondrial ROS levels and cell apoptosis in vitro. Moreover, SOX2OT overexpression also reduced serum creatinine, BUN, urinary ACR, 8‐OHdG, renal tubular injury markers KIM1 and NGAL, ROS levels, and cell apoptosis in vivo. In addition, SOX2OT promoted SIRT1 expression by suppressing its ubiquitination. Besides, interference with SIRT1 reversed the inhibitory effect of SOX2OT overexpression on HG‐induced oxidative stress and apoptosis. Forkhead box A2 (Foxa2) levels were up‐regulated in HG‐cultured HK‐2 cells. Foxa2 could bind to the SOX2OT promoter and suppress its expression. Furthermore, interfering with SOX2OT reversed the inhibitory effect of Foxa2 interference on HG‐induced oxidative stress and apoptosis. Conclusion: Foxa2‐mediated SOX2OT up‐regulation reduced oxidative stress and apoptosis of RTECs by promoting SIRT1 expression, thus alleviating the progression of DN. Summary at a Glance: Renal tubule injury plays a major role in the progression of diabetic nephropathy (DN). This study explored the pathogenesis of DN from the perspective of renal tubular epithelial cell injury mediated by oxidative stress under the effect of high glucose (HG). Authors revealed that lncRNA SOX2OT was lowly expressed in renal tubular epithelial cells; Foxa2 was highly expressed in the DN mouse model and HG‐induced renal tubular epithelial cells; lncRNA SOX2OT promoted SIRT1 expression by suppressing its ubiquitination; Foxa2 could bind to the lncRNA SOX2OT promoter and suppress its expression; overexpression of lncRNA SOX2OT inhibits oxidative stress and apoptosis by up‐regulating the SIRT1 levels and improves renal function. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the role and underlying mechanism of miR‐135b in high glucose‐induced oxidative stress of renal tubular epithelial cells. Here, in vivo experiments found that compared to the control group, miR‐135b expression was significantly up‐regulated in the diabetes group, whereas BMP7 mRNA and protein levels were down‐regulated. In high glucose‐treated renal tubular epithelial cells (HK‐2) in vitro, oxidative stress was induced, which up‐regulated miR‐135b expression. In addition, the regulation of miR‐135b on BMP7 expression was confirmed in HK‐2 cells. Under high glucose conditions, oxidative stress promoted the apoptosis of HK‐2 cells through the up‐regulation of miR‐135b expression. In vivo experiments indicated that interference with miR‐135b improved renal function in mice with diabetic nephropathy. In conclusion, these results indicated that the up‐regulation of miR‐135b expression induced by oxidative stress promotes the apoptosis of HK‐2 cells under high glucose conditions. [ABSTRACT FROM AUTHOR]

Purpose: The present study aimed to investigate whether cervical vagal nerve stimulation (VNS) could prevent retinal ganglion cell (RGC) loss and retinal dysfunction after ischemia/reperfusion (I/R) injury. Methods: First, rats were randomly divided into sham group (n = 4) and VNS group (n = 12). Activation of the nodose ganglia (NOG), nucleus of the solitary tract (NTS), superior salivatory nucleus (SSN), and pterygopalatine ganglion (PPG) neural circuit were evaluated by c-fos expression at 0 h after sham VNS and at 0 h (n = 4), 6 h (n = 4), 72 h (n = 4) after VNS. Secondly, rats were randomly assigned to I/R group (pressure-induced retinal ischemia for 1 h and reperfusion for 1 h in the right eye, n = 16) and I/R+VNS group (right cervical VNS for 2 h during the I/R period, n = 16). The left eye of each rat served as a control. Electroretinogram (ERG), RGC numbers, tumor necrosis factor-α (TNF-α) and vasoactive intestinal polypeptide (VIP) levels in retina were determined. Additionally, the level of VIP in PPG was evaluated. Results: In the first part of the study, compared with the sham group, the VNS group exhibited significantly increased expression of c-fos in NOG, NTS, SSN, and PPG tissues at 0, 6, and 72 h. In the second part of the study, compared with left eyes, retinal function in right eyes (as assessed by the a-wave, b-wave and the oscillatory potential amplitudes of ERG and RGC data) was significantly decreased by I/R. The decreased retinal function was attenuated by VNS. In addition, I/R induced an increase in inflammation, which was reflected by elevated TNF-α expression in the retina. VNS significantly attenuated the increase in I/R-induced inflammation. Moreover, VIP expression in the retina and PPG, which may contribute to the inhibition of the inflammatory response, was significantly increased after VNS. Conclusion: VNS could protect against retinal I/R injury by downregulating TNF-α. Upregulation of VIP expression due to activation of the NOG-NTS-SSN-PPG neural circuit may underlie to the protective effects of VNS. [ABSTRACT FROM AUTHOR]

Highlights • AC3 is predominantly expressed in the cytoplasm of small to medium-sized DRG neurons. • AC3 is preferentially colocalized with CGRP-containing DRG neurons and their primary afferent terminals. • AC3 expression pattern in DRGs is similar between C57/BL6 J mouse and Sprague Dawley rat. Abstract cAMP (Cyclic Adenosine monophosphate), one of the most highly studied second messengers, is regulated by a family of adenylyl cyclase (AC) enzymes. Type 3 adenylyl cyclase (abbreviated as AC3), a subtype of adenylyl cyclase, is reported to be expressed in cilia in the olfactory and central nervous system and plays an important role in many physiological functions such as olfaction, development. However, expression of AC3 in the dorsal root ganglion (DRG) is not reported. In the present study, using immunohistochemical method, we discovered that AC3 immunoreactivity (IR) is predominantly expressed in the cytoplasm of small to medium sized DRG neurons. Double labelling revealed that the majority of AC3 IR are colocalized with CGRP (a peptidergic nociceptor marker), rarely with NF200 (a myelinated neuronal marker) or IB4 (a nonpeptidergic nociceptor marker). Furthermore, dense AC3 IR exists in the superficial dorsal horn, especially in laminaⅠand dorsal part of lamina II, where CGRP-positive DRG neurons terminate. The expression pattern of AC3 is similar between C57/BL6 J mouse and Sprague Dawley rat. For instance, AC3 is primarily expressed in the cell bodies of small to medium sized DRG neurons and the majority of AC3 IR is also in CGRP-containing neurons in rat. Taken together, our data suggest that AC3 is primarily expressed in the small to medium sized cell bodies and central terminals of CGRP-positive DRG neurons, implying AC3 enzyme might potentially function in nociception. [ABSTRACT FROM AUTHOR]

Electroacupuncture (EA) can relieve various pains. However, its mechanism in terms of the transcriptome is still not well-known. To explore the full profile of EA-induced molecular modification in the central nerve system, three twins of goats were selected for a match-paired experiment: EA stimulation (60 Hz, 30 min) and none-EA (control). Goats in the EA group showed an increased (p < 0.05) nociceptive threshold compared with the control goats. Experimental goats were sacrificed at 4 h of the experiment, and the periaqueductal grays were harvested for RNA sequencing. As a result, 2651 differentially expressed genes (1803 up-regulated and 848 down-regulated genes) were found and enriched in 30 Kyoto Encyclopedia of Genes and Genomes pathways and 149 gene ontology terms. EA-regulated five neuropeptide genes (proenkephalin, proopiomelanocortin, preprodynorphin, diazepam-binding inhibitor and proprotein convertase 1 inhibitor) were validated with quantitative PCR. Furthermore, up-regulated glutamate receptors, glutamate transporters, -aminobutyric acid (GABA) receptors, GABA transporters, synaptotagmins or mitogen-activated protein kinase (MAPK) genes might contribute to EA-induced analgesia through regulating the glutamatergic synapse, GABAergic synapse, MAPKs, ribosome or ubiquitin-proteasome pathways. Our findings reveal a full profile of molecular modification in response to EA and provide a solid experimental framework for exploring the mechanisms underlying EA-induced analgesia. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinases (MAPKs), especially p38MAPK, play a pivotal role in chronic pain. Electroacupuncture (EA) relieves inflammatory pain underlying the descending pathway, that is, the periaqueductal gray (PAG), the rostral ventromedial medulla (RVM), and the spinal cord dorsal horn (SCDH). However, whether EA antagonizes inflammatory pain through regulation of p38MAPK in this descending facilitatory pathway is unclear. Complete Freund’s adjuvant (CFA) was injected into the hind paw of rats to establish inflammatory pain model. EA was administrated for 30 min at Zusanli and Kunlun acupoints at 0.5, 24.5, 48.5, and 72.5 h, respectively. The paw withdrawal threshold (PWT), paw edema, and Phosphor-p38MAPK-Immunoreactivity (p-p38MAPK-IR) cells were measured before (0 h) and at 1, 3, 5, 7, 25, and 73 h after CFA or saline injection. EA increased PWT at 1, 3, 25, and 73 h and inhibited paw edema at 25 and 73 h after CFA injection. Moreover, the increasing number of p-p38MAPK-IR cells which was induced by CFA was suppressed by EA stimulation in PAG and RVM at 3 and 5 h and in SCDH at 5, 7, 25, and 73 h. These results suggest that EA suppresses inflammation-induced hyperalgesia probably through inhibiting p38MAPK activation in the descending facilitatory pathway. [ABSTRACT FROM AUTHOR]

To investigate analgesic neural circuits activated by electroacupuncture (EA) at different sets of acupoints in the brain, goats were stimulated by EA at set of Baihui-Santai acupoints or set of Housanli acupoints for 30 min. The pain threshold was measured using the potassium iontophoresis method. The levels of c-Fos were determined with Streptavidin-Biotin Complex immunohistochemistry. The results showed pain threshold induced by EA at set of Baihui-Santai acupoints was 44.74%±4.56% higher than that by EA at set of Housanli acupoints (32.64%±5.04%). Compared with blank control, EA at two sets of acupoints increased c-Fos expression in the medial septal nucleus (MSN), the arcuate nucleus (ARC), the nucleus amygdala basalis (AB), the lateral habenula nucleus (HL), the ventrolateral periaqueductal grey (vlPAG), the locus coeruleus (LC), the nucleus raphe magnus (NRM), the pituitary gland, and spinal cord dorsal horn (SDH). Compared with EA at set of Housanli points, EA at set of Baihui-Santai points induced increased c-Fos expression in AB but decrease in MSN, the paraventricular nucleus of the hypothalamus, HL, and SDH. It suggests that ARC-PAG-NRM/LC-SDH and the hypothalamus-pituitary may be the common activated neural pathways taking part in EA-induced analgesia at the two sets of acupoints. [ABSTRACT FROM AUTHOR]

To investigate patterns of c-Fos and c-Jun expression induced by different frequencies of electroacupuncture (EA) in the brain, goats were stimulated by EA of 0, 2, 60, or 100 Hz at a set of “Baihui, Santai, Ergen, and Sanyangluo” points for 30 min. The pain threshold was measured using the potassium iontophoresis method. The levels of c-Fos and c-Jun were determined with Streptavidin-Biotin Complex immunohistochemistry. The results showed that the pain threshold induced by 60 Hz was 82.2% higher (P<0.01) than that by 0, 2, or 100 Hz (6.5%, 35.2%, or 40.9%). EA induced increased c-Fos and c-Jun expression in most analgesia-related nuclei and areas in the brain. Sixty Hz EA increased more c-Fos or c-Jun expression than 2 Hz or 100 Hz EA in all the measured nuclei and areas except for the nucleus accumbens, the area septalis lateralis, the caudate nucleus, the nucleus amygdala basalis, and the locus coeruleus, in which c-Fos or c-Jun expressions induced by 60 Hz EA did not differ from those by 2 Hz or 100 Hz EA. It was suggested that 60 Hz EA activated more extensive neural circuits in goats, which may contribute to optimal analgesic effects. [ABSTRACT FROM AUTHOR]

The lack of carbon sources severely inhibits denitrification in wastewater with a low C/N ratio. Corncob and rice straw were chosen as supplementary carbon sources to bring into the wetland system to supplement the carbon sources needed for denitrification, and the enhancing effects of the two carbon sources on nitrogen removal from the wetland were studied. The cumulative release of carbon was in the order of rice straw[(145.17±9.44) mg·g -1 ]>corncob[(57.41±5.04) mg·g -1 ] based on the 11-day pure water extraction and release experiment, whereas the cumulative release of nitrogen was in the order of rice straw[(2.31±0.09) mg·g -1 ]>corncob[(0.66±0.08) mg·g -1 ]. The average carbon/nitrogen ratios released and accumulated by corncob and rice straw during the observation period were 94.78 and 63.64, respectively. Corncob was more suited as an additional carbon source than rice straw. COD concentrations in the effluent from the corncob and straw constructed wetlands were found to be below 50 mg·L -1 for the 58-day pilot test of subsurface flow constructed wetlands, except on days 8 to 12. The NO 3 - -N removal rates of the corncob-added built wetlands were 93%-99% over the observation period, with good denitrification performance. In comparison, the lowest NO 3 - -N removal rate of the constructed wetland with the addition of rice straw was only 76.8% at the late stage of operation, and the denitrification rate dropped dramatically. The control group removal rates of NO 3 - -N were only 76.2%-77.7%, indicating a clear lack of carbon sources. The accumulation of NO 2 - -N was also induced by a lack of carbon supply. NO 2 - -N effluent concentrations were 2.5-6 times and 6-26 times higher in the constructed wetlands with rice straw and the control groups, respectively, than those in the wetlands constructed with corncob. The addition of corncob resulted in a more substantial reduction in NO 2 - -N content in the constructed wetland than the addition of rice straw ( P <0.05). The TN removal rates of wetlands constructed with corncob and rice straw and the control group were 83.75%-93.49%, 76.59%-78.85%, and 67.85%-72.56%, respectively, with significant differences among the three ( P <0.01). Finally, pretreatment with dilute alkali heating raised the cumulative carbon release of corncob to (93.73±17.49) mg·g -1 and the carbon/nitrogen ratio to 175.8, significantly improving the carbon release performance of corncob and demonstrating that it is a suitable source of extra carbon.

Lipotoxicity is a recognized pathological trigger and accelerator of nonalcoholic steatohepatitis (NASH). However, the molecular basis of lipotoxicity-induced NASH remains elusive. Here, we systematically mapped the changes in hepatic transcriptomic landscapes in response to lipotoxic insults across multiple species. Conserved and robust activation of the arachidonic acid pathway, in particular the arachidonate 12-lipoxygenase ( ALOX12 ) gene, was closely correlated with NASH severity in humans, macaques with spontaneously developed NASH, as well as swine and mouse dietary NASH models. Using gain- and loss-of-function studies, we found that ALOX12 markedly exacerbated NASH in both mice and Bama pig models. ALOX12 was shown to induce NASH by directly targeting acetyl-CoA carboxylase 1 (ACC1) via a lysosomal degradation mechanism. Overall, our findings reveal a key molecular driver of NASH pathogenesis and suggest that ALOX12-ACC1 interaction may be a therapeutic target in NASH.

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease and has become a leading indication for liver transplantation in the United States. The development of effective therapies for NASH is a major unmet need. Here, we identified a small molecule, IMA-1, that can treat NASH by interrupting the arachidonate 12-lipoxygenase (ALOX12)–acetyl-CoA carboxylase 1 (ACC1) interaction. IMA-1 markedly blocked diet-induced NASH progression in both male mice and Cynomolgus macaque therapeutic models. The anti-NASH efficacy of IMA-1 was comparable to ACC inhibitor in both species. Protein docking simulations and following functional experiments suggested that the anti-NASH effects of IMA-1 were largely dependent on its direct binding to a pocket in ALOX12 proximal to its ACC1-interacting surface instead of inhibiting ALOX12 lipoxygenase activity. IMA-1 treatment did not elicit hyperlipidemia, a known side effect of direct inhibition of ACC enzymatic activity, in both mice and macaques. These findings provide proof of concept across multiple species for the use of small molecule–based therapies for NASH.

As the problem of global warming becomes increasingly serious, the greenhouse gas (GHG) emission reduction measures of constructed wetlands (CWs) have drawn significant attention. Ferric-carbon micro-electrolysis exhibits an excellent effect on wastewater purification as well as the potential to reduce GHG emissions. Therefore, to explore the impact of ferric-carbon micro-electrolysis on GHG emissions from intermittent aeration constructed wetlands, four kinds of different wetlands with different fillers were constructed. The four fillers were ferric-carbon micro-electrolysis filler+gravel (CW-Ⅰ), ferric-carbon micro-electrolysis filler+zeolite (CW-Ⅱ), zeolite (CW-Ⅲ), and gravel (CW-Ⅳ). Intermittent aeration technology was used to aerate the wetland systems. The results show that ferric-carbon micro-electrolysis significantly improved the nitrogen removal efficiency of the intermittent aeration constructed wetlands and reduced GHG emissions. Compared with CW-Ⅳ, the CH 4 fluxes of CW-Ⅰ, CW-Ⅱ, and CW-Ⅲ decreased by 32.81% ( P <0.05), 52.66% ( P <0.05), and 54.50% ( P <0.05), respectively. Among them, zeolite exhibited a stronger reduction effect on CH 4 emissions in both the aeration and non-aeration sections. The ferric-carbon micro-electrolysis substantially reduced N 2 O emissions. In comparison with CW-Ⅳ, CW-, and CW-Ⅱ achieved N 2 O emission reduction by 30.29%-60.63% ( P <0.05) and 43.10%-73.87% ( P <0.05), respectively. During a typical hydraulic retention period, the comprehensive GWP caused by CH 4 and N 2 O emitted by each group of wetland system are (85.21±6.48), (49.24±3.52), (127.97±11.44), and (137.13±11.45) g·m -2 , respectively. The combined use of ferric-carbon micro-electrolysis and zeolite effectively reduces GHG emissions in constructed wetlands. Overall, ferric-carbon micro-electrolysis combined with zeolite (CW-Ⅱ) can be regarded as one of the valuable filler combination methods for constructed wetlands, which can ensure high removal efficiency of pollutants and effective GHG emission reduction in constructed wetlands.

Investigate the effects of plastic film mulching on CH 4 and N 2 O emissions from a vegetable field, a one-year in situ field observation was conducted using a static opaque chamber in a pepper-radish cropping system at the Key Field Station for Monitoring of Eco-Environment of Purple Soil of the Ministry of Agriculture of China at Southwest University, Chongqing. Two treatments (conventional and film mulching) were used to study the influence of film mulching on CH 4 and N 2 O emissions. The results showed that mulching significantly increased the annual average soil pH ( P <0.01), annual surface and subsurface (5 cm) temperature ( P <0.05), and soil moisture content during the radish-growing season ( P <0.05). The mulching also significantly reduced CH 4 emissions in the field ridges ( P <0.05); the average CH 4 flux from ridges during the pepper-growing season was 0.110 mg·(m 2 ·h) -1 and 0.028 mg·(m 2 ·h) -1 , and 0.011 mg·(m 2 ·h) -1 and -0.019 mg·(m 2 ·h) -1 during the radish-growing season, under the conventional and film mulching treatments, respectively. However, across the entire experiment, CH 4 flux from field furrows was not significantly different between the two mulching treatments ( P >0.05), with mean flux values during the pepper-growing season of 0.058 mg·(m 2 ·h) -1 and 0.057 mg·(m 2 ·h) -1 , and 0.083 mg·(m 2 ·h) -1 and 0.092 mg·(m 2 ·h) -1 during the radish-growing season, for conventional and plastic film mulching, respectively. Except for the conventional treatment during the pepper-growing season, CH 4 emissions from ridges were significantly higher than from furrows, but for other treatments, including conventional and film mulching treatments during radish-growing season and film mulching treatment during the pepper-growing season, the CH 4 emissions from furrows were all significantly higher than those from ridges. This was related to the stable anoxic environment created in furrows under high rainfall conditions in Southwest China. The N 2 O emission flux from the ridges during the pepper-growing season was 65.41 μg·(m 2 ·h) -1 and 68.39 μg·(m 2 ·h) -1 under the conventional and film mulching treatments, respectively, and the N 2 O emission flux during the radish-growing season was 78.43 μg·(m 2 ·h) -1 and 66.19 μg·(m 2 ·h) -1 , respectively. The N 2 O flux between conventional treatment and film mulching treatment in ridges or furrows were not significantly different ( P >0.05), while the N 2 O emissions from the ridges were significantly higher than that from the furrows. CH 4 emission flux was significantly positively correlated with surface and subsurface temperature, while N 2 O emission flux was only significantly positively correlated with alkaline nitrogen and ammonium nitrogen content.

Background: Leukocyte telomere has been shown to be related to insulin resistance-related diseases, such as type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). This cross-sectional study investigated the association of leukocyte telomere length (LTL) with NAFLD in T2DM patients., Methods: Clinical features were collected and LTL was measured by Southern blot-based terminal restriction fragment length analysis in 120 T2DM patients without NAFLD and 120 age-matched T2DM patients with NAFLD. NAFLD was clinically defined by manifestations of ultrasonography. The correlation between LTL and clinical and biochemical parameters were analyzed by Pearson correlation or Spearman correlation analysis. Factors for NAFLD in T2DM patients were identified using multiple logistic regressions., Results: LTL in T2DM patients with NAFLD were significantly longer than those without NAFLD (6400.2 ± 71.8 base pairs [bp] vs. 6023.7 ± 49.5 bp, P < 0.001), especially when diabetes duration was less than 2 years. Meanwhile, the trend of shorter LTL was associated with the increased diabetes duration in T2DM patient with NAFLD, but not in T2DM patients without NAFLD. Finally, LTL (odds ratio [OR]: 1.001, 95% confidence interval [CI]: 1.000-1.002, P = 0.001), as well as body mass index (OR: 1.314, 95% CI: 1.169-1.477, P < 0.001) and triglycerides (OR: 1.984, 95% CI: 1.432-2.747, P < 0.001), had a significant association with NAFLD status in T2DM patients., Conclusions: T2DM patients with NAFLD had a significantly longer LTL than those without NAFLD. The longer LTL was especially evident in the early stage of T2DM, indicating that longer LTL may be used as a biomarker for NAFLD in T2DM patients.

Increasing evidence suggests that T cells and glia participate in the process of neuropathic pain. However, little is known about the involvement of T cells or the interaction between glia and T cells at the molecular level. Here we investigated the phenotype of T cell infiltration into the spinal cord in inflammatory pain and explored potential crosstalk between glia and T cells. The establishment of monoarthritis produced T cell infiltration and astrocyte activation, exhibiting similar kinetics in the spinal cord. T-cell-deficient (Rag1 -/- ) mice significantly attenuated MA-induced mechanical allodynia and GFAP upregulation. Double immunofluorescence staining showed that CD3 mainly colocalized with interferon-gamma (IFN-γ). Western blot and flow cytometry showed that multiple intrathecal administrations of astrocytic inhibitor fluorocitrate decreased IFN-γ-production without decreasing T cell number in the spinal cord. Spinal IFN-γ blockade reduced MA-induced mechanical allodynia and astroglial activation. In contrast, treatment with rIFN-γ directly elicited persistent mechanical allodynia and upregulation of GFAP and pJNK1/2 in naïve rats. Furthermore, rIFN-γ upregulated the phosphorylation of NF-κB p65 in cultured astrocytes vitro and spinal dorsal horn vivo. The results suggest that Th1 cells and astrocytes maintain inflammatory pain and imply that there may be a positive feedback loop between these cells via IFN-γ.

Electroacupuncture (EA) can relieve various pains. However, its mechanism in terms of the transcriptome is still not well-known. To explore the full profile of EA-induced molecular modification in the central nerve system, three twins of goats were selected for a match-paired experiment: EA stimulation (60 Hz, 30 min) and none-EA (control). Goats in the EA group showed an increased ( p < 0.05) nociceptive threshold compared with the control goats. Experimental goats were sacrificed at 4 h of the experiment, and the periaqueductal grays were harvested for RNA sequencing. As a result, 2651 differentially expressed genes (1803 up-regulated and 848 down-regulated genes) were found and enriched in 30 Kyoto Encyclopedia of Genes and Genomes pathways and 149 gene ontology terms. EA-regulated five neuropeptide genes ( proenkephalin , proopiomelanocortin , preprodynorphin , diazepam-binding inhibitor and proprotein convertase 1 inhibitor ) were validated with quantitative PCR. Furthermore, up-regulated glutamate receptors, glutamate transporters, γ-aminobutyric acid ( GABA ) receptors, GABA transporters, synaptotagmins or mitogen-activated protein kinase ( MAPK ) genes might contribute to EA-induced analgesia through regulating the glutamatergic synapse, GABAergic synapse, MAPKs, ribosome or ubiquitin-proteasome pathways. Our findings reveal a full profile of molecular modification in response to EA and provide a solid experimental framework for exploring the mechanisms underlying EA-induced analgesia., Competing Interests: The authors declare no conflict of interest.

In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (δ-, μ-, and κ-receptor) induced by electroacupuncture (EA) in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated (P < 0.01) with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.