Blockade of PI3K/AKT signaling pathway by Astragaloside IV attenuates ulcerative colitis via improving the intestinal epithelial barrier.

BACKGROUND: The specific pathogenesis of UC is still unclear, but it has been clear that defects in intestinal barrier function play an important role in it. There is a temporary lack of specific drugs for clinical treatment. Astragaloside IV (AS-IV) is one of the main active ingredients extracted from Astragalus root and is a common Chinese herbal medicine for the treatment of gastrointestinal diseases. This study aimed to determine whether AS-IV has therapeutic value for DSS or LPS-induced intestinal epithelial barrier dysfunction in vivo and in vitro and its potential molecular mechanisms. METHODS: The intestinal tissues from UC patients and colitis mice were collected, intestinal inflammation was observed by colonoscopy, and mucosal barrier function was measured by immunofluorescence staining. PI3K/AKT signaling pathway activator YS-49 and inhibitor LY-29 were administered to colitic mice to uncover the effect of this pathway on gut mucosal barrier modulation. Then, network pharmacology was used to screen Astragaloside IV (AS-IV), a core active component of the traditional Chinese medicine Astragalus membranaceus. The potential of AS-IV for intestinal barrier function repairment and UC treatment through blockade of the PI3K/AKT pathway was further confirmed by histopathological staining, FITC-dextran, transmission electron microscopy, ELISA, immunofluorescence, qRT-PCR, and western blotting. Finally, 16 S rRNA sequencing was performed to uncover whether AS-IV can ameliorate UC by regulating gut microbiota homeostasis. RESULTS: Mucosal barrier function was significantly damaged in UC patients and murine colitis, and the activated PI3K/AKT signaling pathway was extensively involved. Both in vivo and vitro showed that the AS-IV-treated group significantly relieved inflammation and improved intestinal epithelial permeability by inhibiting the activation of the PI3K/AKT signaling pathway. In addition, microbiome data found that gut microbiota participates in AS-IV-mediated intestinal barrier recovery as well. CONCLUSIONS: Our study highlights that AS-IV exerts a protective effect on the integrality of the mucosal barrier in UC based on the PI3K/AKT pathway, and AS-IV may serve as a novel AKT inhibitor to provide a potential therapy for UC.

Deficiency of microRNA-10b promotes DSS-induced inflammatory response via impairing intestinal barrier function.

Inflammatory bowel disease (IBD) is a non-specific inflammatory disease of the intestine with the pathogenesis to be largely unknown. We found that microRNA (miR)-10b knock-out mice displayed mild IBD symptoms, suggesting that miR-10b may be involved in the onset and development of IBD. This study focuses on elucidating the role of miR-10b in IBD. The colitis model was induced by feeding the mice with 2.5% dextran sodium sulfate (DSS), and the expression levels of miR-10b in colon tissue and blood samples were examined. The severity of colitis was assessed by disease activity index, colon length, histopathological damage, intestinal permeability and ELISA. Then, after transfection of Caco-2 cells with miR-10b mimic and inhibitor, qRT-PCR was used to detect the expression levels of intestinal barrier related genes in colon tissues and cells. miR-10b levels were significantly reduced in mice with DSS-induced acute colitis. Compared with wild-type (WT) mice, miR-10b knockout mice were more sensitive to DSS-induced colitis characterized by increased inflammatory cell infiltration and more severe disruption of colonic barrier function. In addition, by inhibiting miR-10b and thus increasing intestinal barrier gene expression in Caco-2 cells, we found that miR-10b suppressed inflammatory responses and enhanced intestinal barrier function both in vivo and in vitro. miR-10b inhibits the inflammatory response in DSS-induced acute colitis mice in vivo and enhances intestinal barrier function in vitro, suggesting that miR-10b plays a key role in the developmental process of IBD. Thus, miR-10b may be expected to be a new target for the treatment of IBD.

HPLC-DVD combined with chemometrics to analyze the correlation between the Q-marker content and color of Corni Fructus.

Although Corni Fructus (CF) is a fruit with great economic value and development potential in medicine and food, too much reliance on personal experience for quality evaluation seriously limits the trading and circulation of CF. In the present study, through the research on the correlation between the chemical composition and the appearance color, a standard colorimetric card related to CF quality was established, which simplified the quality evaluation process and improved the accuracy of the visual evaluation of CF. Firstly, a total of 29 batches of CF from different places were collected. Then, "imread" in the MATLAB software was used to convert the color of all samples into RGB values, and HPLC-DVD was used to measure the content of the main chemical components in CF. Thereafter, the correlation between the content and color was studied by using MLR, BP-ANNs and SVM chemometric tools to screen the Q-marker of CF, which was further confirmed by in vivo and in vitro experiments. Finally, the Q-marker standard colorimetric card with the best fitting degree is established according to the prediction model. Thus, this study provides an auxiliary reference for the color evaluation of CF and a reference for the standardization and quantification of the macro characteristics of traditional Chinese medicine and food.

IL-6 induced enhanced clearance of proANP and ANP by insulin-degrading enzyme in T1DM mice.

Cardiovascular disease (CVD) is a prevalent cause of morbidity and mortality in type I diabetes mellitus (T1DM). However, the pathophysiological mechanisms underlying the relationship between CVD, CVD risk factors, and T1DM have not yet been sufficiently explored. Here, we report that insulin-degrading enzyme (IDE) effectively degrades the precursor of atrial natriuretic peptide (proANP) in HEK293T cells. The pro-inflammatory cytokine IL-6 elicited a significant dose-dependent increase in IDE protein expression. Inhibition of the ERK/MAPK signaling pathway with selumetinib abolished the IL-6-stimulated increase in IDE protein levels and decreased ANP secretion in H9C2 cells. Importantly, the T1DM mouse model displayed lower proANP in the heart and ANP in serum, due to increased IDE expression and activity. Our results suggest a novel role of IL-6 in ANP metabolism via IDE and provide possibilities for new potential therapeutic strategies for diabetes-related cardiovascular complications.

Ultrasound-assisted enzymatic extraction of Corni Fructus alpha-glucosidase inhibitors improves insulin resistance in HepG2 cells.

Corni Fructus (CF) is a traditional medicine and beneficial food with multifaceted protective effects against diabetes and its complications. Since alpha-glucosidase inhibitors (GIs) are promising first-choice oral antihyperglycemic drugs for diabetes, we examined whether GIs from CF (GICF) are useful for diabetes treatment. Therefore, GICF was extracted by ultrasound-assisted enzymatic extraction (UAEE) that is optimized by a three-level, four-factor Box-Behnken design and determined by ultra-performance liquid chromatography. Compared to 36.31 mg g-1 without enzyme treatment, the GICF yield increased to 70.44 mg g-1via UAEE under optimum conditions (0.5% compound enzyme extracted in 23 min at 46 °C and pH 4.8). The activity (91.99%) of GICF was as predicted (93.28%). When GICF was used in an insulin-resistant HepG2 cell model, it significantly ameliorated the glucose metabolism in a dose-dependent manner. Our findings indicate that UAEE may be an innovative method for functional food extraction and a potential strategy for high-quality food ingredient (such as GI) production with high efficiency and productivity.

Total Saponins Isolated from Corni Fructus via Ultrasonic Microwave-Assisted Extraction Attenuate Diabetes in Mice.

Saponins have been extensively used in the food and pharmaceutical industries because of their potent bioactive and pharmacological functions including hypolipidemic, anti-inflammatory, expectorant, antiulcer and androgenic properties. A lot of saponins-containing foods are recommended as nutritional supplements for diabetic patients. As a medicine and food homologous material, Corni Fructus (CF) contains various active ingredients and has the effect of treating diabetes. However, whether and how CF saponins attenuate diabetes is still largely unknown. Here, we isolated total saponins from CF (TSCF) using ultrasonic microwave-assisted extraction combined with response surface methodology. The extract was further purified by a nonpolar copolymer styrene type macroporous resin (HPD-300), with the yield of TSCF elevated to 13.96 mg/g compared to 10.87 mg/g obtained via unassisted extraction. When used to treat high-fat diet and streptozotocin-induced diabetic mice, TSCF significantly improved the glucose and lipid metabolisms of T2DM mice. Additionally, TSCF clearly ameliorated inflammation and oxidative stress as well as pancreas and liver damages in the diabetic mice. Mechanistically, TSCF potently regulated insulin receptor (INSR)-, glucose transporter 4 (GLUT4)-, phosphatidylinositol 3-kinase (PI3K)-, and protein kinase B (PKB/AKT)-associated signaling pathways. Thus, our data collectively demonstrated that TSCF could be a promising functional food ingredient for diabetes improvement.

Protective Effects of Iridoid Glycoside from Corni Fructus on Type 2 Diabetes with Nonalcoholic Fatty Liver in Mice.

Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease. Accumulating evidence has demonstrated that nonalcoholic fatty liver disease (NAFLD) shares common typical features with T2DM, and they affect each other extensively. Thus, NAFLD has emerged as a novel target for T2DM prevention and care. Although Corni Fructus (CF) and its extracts have a therapeutic effect on T2DM, its effects and mechanisms on T2DM with NAFLD are far from elucidated. In this study, a mouse model of T2DM with NAFLD complication was established in ICR mice by feeding a high-fat, high-sugar (HFHS) diet and intraperitoneally injecting with a low dose of streptozotocin (STZ). Then, the effects of iridoid glycosides (IG) extracted from CF on this mouse model were investigated. We found that 4-week IG administration remarkably alleviated hyperglycemia and insulin resistance and significantly reduced inflammation, oxidative stress, and fat accumulation in the liver of T2DM with NAFLD mice. Further studies showed that IG inhibited the NF-κB but enhanced the PI3K-AKT signaling pathway. In summary, these results indicated that the IG from CF has potential therapeutic effects on T2DM with NAFLD.

Ultrasonic-microwave assisted extraction of total triterpenoid acids from Corni Fructus and hypoglycemic and hypolipidemic activities of the extract in mice.

Triterpene acids, the main component of Corni Fructus, could improve diabetes mellitus, for which the underlying hypoglycemic mechanism is still unclear, in patients. In this study, total triterpenoid acids were extracted by ultrasonic-microwave assisted extraction optimized by the response surface methodology. The extract was then purified with an X-5 macroporous resin, and the yield of total triterpenoid acids increased to 281.24 mg g-1 as compared with the 35.71 mg g-1 obtained by unassisted extraction. The contents of five components were determined by ultrafast performance liquid chromatography. In addition, the hypoglycemic and hypolipidemic activities of total triterpenoid acids in diabetic mice induced by streptozotocin and a high fat diet were studied. The results indicated that all parameters (oral glucose tolerance, insulin resistance and liver damage) related to diabetes were significantly improved by total triterpenoid acids. Furthermore, total triterpenoid acids significantly recovered the expression level of AMP-activated protein kinase and its downstream proteins, including acetyl-CoA carboxylase, carnitine palmityltransferase-1, peroxisome proliferator-activated receptor alpha, sterol regulatory element-binding protein 1c and fatty acid synthase. Altogether, total triterpenoid acids could ameliorate hyperlipidemia and hyperglycemia in diabetic mice, probably by activating the AMP-activated protein kinase-peroxisome proliferator-activated receptor signaling pathway and inhibiting the sterol regulatory element-binding protein 1c and fatty acid synthase signaling pathways. Therefore, total triterpene acids, isolated from Corni Fructus which is a prevailing health food, could be a functional food ingredient with therapeutic and commercial values.

Corni Fructus as a Natural Resource Can Treat Type 2 Diabetes by Regulating Gut Microbiota.

Accumulating evidence suggests that gut microbiota plays a crucial role in the development of metabolic diseases, especially type 2 diabetes mellitus (T2DM). The nutrient-rich resource Cornus Fructus (CF) showed curative effects on diabetes mellitus. However, the mechanism underlying its hyperglycemic activity remains obscure. Herein, the antidiabetic potential of four extracts from CF, including saponin (CTS), iridoid glycoside (CIG), tannin (CT), and alcohol extract (CCA) was evaluated in vivo. The results showed that all four extracts could increase the body weight, decrease the blood glucose levels, and elevate the glucose tolerance. Moreover, insulin sensitivity and lipid profile were significantly improved in fed mice. In the [Formula: see text]-diversity index of samples, compared to the DM group, the diversity and richness of gut microbiota in mice to a certain extent were reduced in both CF extracts and Metformin (PC). Among them, there was statistical significance in PC (ACE, [Formula: see text]) and CCA (ACE, [Formula: see text]; chao1: [Formula: see text]). Beta diversity showed the same trend as the UPGMA clustering trees, which revealed that CF extracts could improve intestinal homeostasis in T2DM mice. Also, CF extracts could elevate the production of short-chain fatty acids, as well as regulate the composition of gut microbiota. The key bacteria related to T2DM including Firmicutes, Bacteroides, Lactobacillus, and Clostridium were modulated by metformin and CF. Altogether, CF is a potential nutrient-rich candidate that can be used in functional foods for the treatment of T2DM, and the change of gut microbiota might be a novel mechanism underlying its hyperglycemic activity.

Selection of suitable reference genes for qRT-PCR expression analysis of Codonopsis pilosula under different experimental conditions.

Codonopsis pilosula is a well-known medicinal plant. Although its transcriptome sequence has been published, suitable reference genes have not been systematically identified for conducting expression analyses via quantitative real-time polymerase chain reaction (qRT-PCR). To screen appropriate genes for use with this species, we applied four different methods-GeNorm, NormFinder, BestKeeper, and RefFinder-to evaluate the stability of 13 candidates: CpiEF1Bb, CpiCACS, CpiF-Box, Cpiß-Tubulin, CpiGAPDH, CpiActin2, CpiAPT1, CpiActin7, CpiActin8, CpiRPL6, CpiHAF1, CpiTubulin6, and CpiUBQ12. Expression was examined by qRT-PCR for various tissue types, chemical treatments, and developmental stages. For all tested samples, CpiGAPDH proved to be the most stable. Comprehensive analysis indicated that the most stable internal reference genes were CpiF-Box and CpiCACS in different tissues and at different developmental stages, respectively. Under NaCl stress, CpiAPT1 was the best internal reference gene. For methyl jasmonate and abscisic acid treatments, CpiGAPDH and CpiF-Box, respectively, presented the highest degree of expression stability. Based on these findings, we chose CpiSPL9 as the target gene for validating the suitability of these selected reference genes. All of these results provide a foundation for accurate quantification of expression levels by genes of interest in C. pilosula.

Establishment of in vitro culture system for Codonopsis pilosula transgenic hairy roots.

The aim of the study was to establish a reliable system of transgenic hairy roots in Codonopsis pilosula through Agrobacterium-mediated genetic transformation. For this, we optimized several steps in the process of A. rhizogenes strain C58C1 mediated hairy root induction, including the most appropriate medium, explant type, time for infection and co-cultivation. We achieved an induction rate of up to 100% when the roots of C. pilosula seedlings were used as explants, infected with A. rhizogenes C58C1 harboring pCAMBIA1305 for 5 min, followed by induction on 1/2MS supplemented with 0.2 mg/L naphthylacetic acid and 200 mg/L cefotaxime sodium. The co-transformed hairy roots were confirmed by PCR amplification of hygromycin phosphotransferase II gene and histochemical GUS assay, and the efficiency of transformation was 70% and 68.3%, respectively, when no hygromycin selection pressure was exerted. To increase biomass production, we excised and self-propagated the transformed hairy roots, which produce saponins. Our successful establishment of an in vitro culture system of transgenic hairy root for this species lays the foundation not only for assessing gene expression and function but also for obtaining high levels of secondary metabolites through genetic engineering technology.

Comparative Study of Crude and Wine-Processing Corni Fructus on Chemical Composition and Antidiabetic Effects.

Wine processing is a specialized technology which involves sautéing crude herbal medicine using Chinese rice wine. Herein, we identified the changes in chemical profiles and antidiabetic effects of Corni Fructus (CF) after wine processing in high-fat diet (HFD) streptozotocin- (STZ-) induced diabetic mice. A novel high-efficiency method for simultaneously quantifying gallic acid, 5-hydroxymethylfurfural, morroniside, loganin, sweroside, and cornuside by UPLC was developed, and validating crude and wine-processing CF was done for the first time. Mice were randomly divided into the following groups and orally given different solutions for 4 weeks: normal group (NC, 0.4% (w/v) CMC-Na), model group (DM, 0.4% (w/v) CMC-Na), crude CF group (CP, 3.87 g/kg), and wine-processing CF group (PP, 3.87 g/kg) followed by HFD and multiple subcutaneous injection of STZ (40 mg/kg) to induce the diabetes model except the NC group. Biochemical indexes (body weight, fasting blood glucose level, lipid level, insulin, and free fatty acid) and other parameters involving liver toxicity were measured with commercial kits and immunohistochemical method. Comparative studies on pharmacology showed that the crude extracts possess higher efficacy on hypoglycemia and hypolipidemia, while wine-processing products exhibit better effects on liver preservation. Our data suggested that wine processing was recommended when CF was used for protecting the liver; however, crude products should be used as antidiabetic drugs.

SmbHLH37 Functions Antagonistically With SmMYC2 in Regulating Jasmonate-Mediated Biosynthesis of Phenolic Acids in Salvia miltiorrhiza.

Jasmonates (JAs) are integral to various defense responses and induce biosynthesis of many secondary metabolites. MYC2, a basic helix-loop-helix (bHLH) transcription factor (TF), acts as a transcriptional activator of JA signaling. MYC2 is repressed by the JASMONATE ZIM-domain (JAZ) proteins in the absence of JA, but de-repressed by the protein complex SCFCOI1 on perception of JA. We previously reported that overexpression of SmMYC2 promotes the production of salvianolic acid B (Sal B) in Salvia miltiorrhiza. However, the responsible molecular mechanism is unclear. Here, we showed that SmMYC2 binds to and activates the promoters of its target genes SmTAT1, SmPAL1, and SmCYP98A14 to activate Sal B accumulations. SmbHLH37, a novel bHLH gene significantly up-regulated by constitutive expression of SmMYC2, was isolated from S. miltiorrhiza for detailed functional characterization. SmbHLH37 forms a homodimer and interacts with SmJAZ3/8. Overexpression of SmbHLH37 substantially decreased yields of Sal B. SmbHLH37 binds to the promoters of its target genes SmTAT1 and SmPAL1 and blocks their expression to suppress the pathway for Sal B biosynthesis. These results indicate that SmbHLH37 negatively regulates JA signaling and functions antagonistically with SmMYC2 in regulating Sal B biosynthesis in S. miltiorrhiza.

Hypoglycemic, hypolipidemic and antioxidant effects of iridoid glycosides extracted from Corni fructus: possible involvement of the PI3K-Akt/PKB signaling pathway.

Iridoid glycosides (CIG) are the major component of Corni fructus. In this work, we researched the antioxidative, hypoglycemic and lowering blood lipids effects of CIG on diabetic mice induced by a high-fat diet (HFD) and streptozotocin (STZ). Furthermore, to investigate the molecular mechanism of action, the phosphorylation and protein expression of phosphoinositide 3-kinase (PI3K) and its downstream proteins, such as insulin receptor (INSR), protein kinase B (Akt/PKB) and glucose transporter 4 (GLUT4) have been detected. The results showed that CIG significantly improved oral glucose tolerance in diabetic mice. Biochemical indices also revealed that CIG had a positive effect on lipid metabolism and oxidative stress. In addition, CIG can significantly enhance the expression level of the PI3K-Akt/PKB pathway related proteins in skeletal muscle, which is the key pathway of insulin metabolism. These findings show that CIG can improve the hyperglycemia and hyperlipidemia of HFD-STZ-induced diabetic mice through the PI3K-Akt/PKB signaling pathway, and CIG might be a potential medicine or functional food for type 2 diabetes mellitus remedies.

Functional Characterization of Selected Universal Stress Protein from Salvia miltiorrhiza (SmUSP) in Escherichia coli.

The multigene universal stress protein (USP) family is evolutionarily conserved. Members play indispensable roles in plant tolerance to abiotic stresses. Although relatively well-characterized in model plants, such as Arabidopsis thaliana and Oryzasativa, this family has not been investigated in Salvia miltiorrhiza, an important herbal plant for which yields can be limited by various abiotic stresses. Here, we identified 32 USP family members in the S. miltiorrhiza genome, and used phylogenetic analysis to sort these SmUSPs into four groups. Groups A and B belong to the ATP-binding class whereas Groups C and D are in the non-ATP-binding class. Motif analysis and multiple sequence alignment hinted that members of group A and B were able to bind ATP. Our qRT-PCR data from different tissues/organs and under salt and heat stresses provided an overall expression pattern for those genes. Three SmUSPs (SmUSP1, SmUSP8, and SmUSP27) were cloned from S. miltiorrhiza and functionally characterized in Escherichiacoli. Compared with the control cells, those that expressed SmUSPs exhibited enhanced tolerance to salt, heat, and a combination of the two. This suggested that the protein has a protective role in cells when exposed to single-stress and multiple-stress conditions. Our findings provide valuable information that helps improve our understanding of the evolutionary and functional conservation and diversity associated with the USP gene family in S. miltiorrhiza.

Transcriptional Responses and Gentiopicroside Biosynthesis in Methyl Jasmonate-Treated Gentiana macrophylla Seedlings.

Gentiana macrophylla, a medicinal plant with significant pharmacological properties, contains the bioactive compound gentiopicroside. Methyl jasmonate (MeJA) is an effective elicitor for enhancing the production of such compounds. However, little is known about MeJA-mediated biosynthesis of gentiopicroside. We investigated this phenomenon as well as gene expression profiles to determine the molecular mechanisms for MeJA-mediated gentiopicroside biosynthesis and regulation in G. macrophylla. Our HPLC results showed that Gentiana macrophylla seedlings exposed to MeJA had significantly higher concentrations of gentiopicroside when compared with control plants. We used RNA sequencing to compare transcriptional profiles in seedlings treated for 5 d with either 0 µmol L-1 MeJA (C) or 250 µmol L-1 MeJA (M5) and detected differentially expressed genes (DEGs). In total, 77,482 unique sequences were obtained from approximately 34 million reads. Of these, 48,466 (57.46%) sequences were annotated based on BLASTs performed against public databases. We identified 5,206 DEGs between the C and M5 samples, including genes related to the α-lenolenic acid degradation pathway, JA signaling pathway, and gentiopicroside biosynthesis. Expression of numerous enzyme genes in the glycolysis pathway was significantly up-regulated. Many genes encoding transcription factors (e.g. ERF, bHLH, MYB, and WRKY) also responded to MeJA elicitation. Rapid acceleration of the glycolysis pathway that supplies precursors for IPP biosynthesis and up-regulates the expression of enzyme genes in that IPP pathway are probably most responsible for MeJA stimulation of gentiopicroside synthesis. Our qRT-PCR results showed that the expression profiles of 12 gentiopicroside biosynthesis genes were consistent with the RNA-Seq data. These results increase our understanding about how the gentiopicroside biosynthesis pathway in G. macrophylla responds to MeJA.

Chemical composition and antimicrobial activity of the essential oils from Chloranthus japonicus Sieb. and Chloranthus multistachys Pei.

We examined the composition and antimicrobial activity of two essential oils from Chloranthus japonicus Sieb. and Chloranthus multistachys Pei. GC-FID and GC-MS analyses identified 48 and 39 compounds, which represented 95.56% and 94.58%, respectively, of all components in these oils. Of these, 28 compounds were common to both, with a relatively high amount of oxygenated monoterpenes (50.95% and 39.97%). Antimicrobial properties were evaluated in vitro via disc diffusion and microbroth dilution assays. Activities were strong against most tested microorganisms, with inhibition zones ranging from 8.1 to 22.2 mm. For both species, minimum values for inhibitory and bactericidal concentrations were 0.39 to 12.50 mg/mL and 0.78 to 50.00 mg/mL, respectively. These results suggest that these essential oils are potent natural sources of antimicrobial agents for the medicinal and pharmaceutical industries.

Alpha-2 adrenoceptor mediating the facilitatory effect of norepinephrine on the glycine response in the spinal dorsal horn neuron of the rat.

Effects of norepinephrine (NE) on the glycine-mediated inhibitory response were investigated in neurons acutely dissociated from the rat spinal dorsal horn, using nystatin perforated patch recording mode under voltage-clamp conditions. NE reversibly and concentration dependently facilitated Cl(-) current induced by 3 x 10(-5) M glycine. NE neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. This effect could be mimicked by clonidine (10(-7) M) and blocked by yohimbine (10(-6) M), respectively. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89), an inhibitor of protein kinase A, effectively mimicked the effect of NE on glycine response, whereas chelerythrine (an inhibitor of protein kinase C) failed. NE further enhanced glycine response even in the presence of chelerythrine or stearoylcarnitine chloride (another inhibitor of protein kinase C) or chelerythrine together with stearoylcarnitine chloride. The present results suggest that alpha2-adrenoceptor is involved in the potentiation of NE on glycine response in freshly isolated spinal dorsal horn neurons. Activation of alpha2-adrenoceptor down-regulates the activity of protein kinase A that results in the potentiation of the glycinergic inhibitory effects within the spinal dorsal horn.

Serotonin potentiation of glycine-activated whole-cell currents in the superficial laminae neurons of the rat spinal dorsal horn is mediated by protein kinase C.

The modulatory effects of serotonin (5-HT) on glycine (Gly)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (I and II) of the rat spinal dorsal horn using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Our results demonstrate that (1). Gly acted on strychnine (STR)-sensitive Gly receptors and elicited inward Cl(-) currents (I(Gly)) at a holding potential of -40 mV; (2). 5-HT potentiated I(Gly) without affecting the reversal potential of I(Gly); (3). the agonist (alpha-methyl-5-HT) and antagonist (ketanserine) of 5-HT(2) receptor mimicked and blocked the potentiating effect of 5-HT on I(Gly), respectively; (4). bisindolylmaleimide I (BIM), a selective inhibitor of protein kinase C (PKC), reduced the potentiating effect of 5-HT on I(Gly); and (5). 5-HT-induced enhancement of I(Gly) was not affected by pretreatment with 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA AM), a Ca(2+) chelator. These results indicate that (1). the potentiation of 5-HT on I(Gly) is mediated by 5-HT(2) receptor and through Ca(2+)-independent PKC intracellular signal transduction pathway; and (2). the interactions between 5-HT and Gly might modulate the transmission of nociceptive information through the spinal cord.