Proto-Oncogene Proteins c-akt/metabolism* ; Butyrates ; Ligands ; Glucose ; Receptors, G-Protein-Coupled/metabolism* ; Glycogen Synthase Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta ; Phosphorylation

Carthamus tinctorius is proved potent in treating ischemic stroke. Flavonoids, such as safflower yellow, hydroxysafflor yellow A(HSYA), nicotiflorin, safflower yellow B, and kaempferol-3-O-rutinoside, are the main substance basis of C. tinctorius in the treatment of ischemic stroke, and HSYA is the research hotspot. Current studies have shown that C. tinctorius can prevent and treat ischemic stroke by reducing inflammation, oxidative stress, and endoplasmic reticulum stress, inhibiting neuronal apoptosis and platelet aggregation, as well as increasing blood flow. C. tinctorius can regulate the pathways including nuclear factor(NF)-κB, mitogen-activated protein kinase(MAPK), signal transducer and activator of transcription protein 3(STAT3), and NF-κB/NLR family pyrin domain containing 3(NLRP3), and inhibit the activation of cyclooxygenase-2(COX-2)/prostaglandin D2/D prostanoid receptor pathway to alleviate the inflammatory development during ischemic stroke. Additionally, C. tinctorius can relieve oxidative stress injury by inhibiting oxidation and nitrification, regulating free radicals, and mediating nitric oxide(NO)/inducible nitric oxide synthase(iNOS) signals. Furthermore, mediating the activation of Janus kinase 2(JAK2)/STAT3/suppressor of cytokine signaling 3(SOCS3) signaling pathway and phosphoinositide 3-kinase(PI3 K)/protein kinase B(Akt)/glycogen synthase kinase-3β(GSK3β) signaling pathway and regulating the release of matrix metalloproteinase(MMP) inhibitor/MMP are main ways that C. tinctorius inhibits neuronal apoptosis. In addition, C. tinctorius exerts the therapeutic effect on ischemic stroke by regulating autophagy and endoplasmic reticulum stress. The present study reviewed the molecular mechanisms of C. tinctorius in the treatment of ischemic stroke to provide references for the clinical application of C. tinctorius.
Carthamus tinctorius/chemistry* ; Chalcone/therapeutic use* ; Cyclooxygenase 2/metabolism* ; Cytokines/metabolism* ; Flavonoids/therapeutic use* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Humans ; Ischemic Stroke/drug therapy* ; Janus Kinase 2/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Phosphatidylinositol 3-Kinase/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Prostaglandin D2 ; Proto-Oncogene Proteins c-akt/metabolism* ; Quinones/pharmacology*

BACKGROUND: Acquired and primary resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is still the bottleneck of clinical treatment of advanced non-small cell lung cancer (NSCLC). STE029 is a novel anticancer drug which consists of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) inhibitor and novel cancer cell membrane targeting molecular. This study aimed to investigate the reversal mechanism of EGFR-TKI resistance by STE029 in lung adenocarcinoma. METHODS: CCK8 test was used to test the cell viability and survival rate of EGFR mutated PC9 cell (Gefitinib sensitive), PC9/BB4 cell (acquired Gefitinib resistant), and EGFR wild type A549 cell after treatment of STE029, Gefitinib or combination of both. EdU test was applied to detect changes in cell cycle and Hoechst 33258 was applied to detect apoptosis rate in overcoming the EGFR-TKI resistance. The activity of EGFR/PI3K/Akt, cell cycle and apoptosis signal pathways were examined. In vivo, nude mice were exposed to STE029, Gefitinib and STE029+Gefitinib for 5 wk. And the the tumor volume was measured and tumor weight was obtained on the last day. RESULTS: (1) PC9 cells was highly sensitive to Gefitinib, while PC9/BB4 and A549 cell showed significant resistance to Gefitinib treatment; (2) STE029+Gefitinib treatment could significantly decrease the 50% inhibitory concentrarion (IC₅₀) of Gefitinib in PC9, PC9/BB4 and A549 cells (P<0.05, respectively); (3) In PC9 and PC9/BB4 cells, STE029+Gefitinib can block cell cycle and inhibit cell proliferation (P<0.001), while there was no significant difference in apoptosis rate among three drug intervention groups (P>0.05); However, apoptosis rate was increased in STE029+Gefitinib group in A549 cell (P<0.01), while no significance detected in cell proliferation (P>0.05). (4) In PC9 and PC9/BB4 cells, the combination of STE029 and Gefitinib could downregulate p-EGFR, p-Akt, p-Cyclin D1 and Cyclin D1 (P<0.001), and upregulate the expression of GSK-3β (P<0.001), and the expression of cleaved caspase-8, caspase-8 cleaved caspase-9, caspase-9 showed no difference among groups (P>0.05). In A549 cells, the combination of STE029 and Gefitinib could downregulate p-Akt (P<0.001) and upregulate cleaved caspase-8 and cleaved caspase-9 (P<0.001); (5)In vivo, the combination of STE029 and Gefitinib effectively inhibited tumor development and progression compared to STE029 alone or Gefitinib alone, with significant difference (P<0.05) in PC9 and PC9/BB4 xenografted tumor. CONCLUSIONS STE029 could sensitize Gefitinib by inhibiting EGFR/PI3K/Akt pathway, blocking the tumor cell cycle and proliferation and inducing apoptosis through caspase-8 and caspase-9 dependent pathway. STE029 deserves further investigations in overcoming EGFR-TKI resistance in lung cancer.
Animals ; Mice ; Humans ; Gefitinib/pharmacology* ; Caspase 9 ; Caspase 8 ; Cyclin D1 ; Carcinoma, Non-Small-Cell Lung ; Glycogen Synthase Kinase 3 beta ; Mice, Nude ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Lung Neoplasms/genetics* ; Adenocarcinoma of Lung/drug therapy* ; Protein Kinase Inhibitors/pharmacology* ; ErbB Receptors/genetics*

Type 1 diabetes mellitus (T1DM)-induced cognitive dysfunction is common, but its underlying mechanisms are still poorly understood. In this study, we found that knockout of conventional protein kinase C (cPKC)γ significantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles, but did not affect the activities of GSK-3β and PP2A in the hippocampal neurons of T1DM mice. cPKCγ deficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice. Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγ deficiency in T1DM mice. Moreover, cPKCγ deficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro. The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor. In conclusion, these results indicated that cPKCγ promotes autophagy through the AMPK/mTOR signaling pathway, thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles.
AMP-Activated Protein Kinases/metabolism* ; Animals ; Autophagy ; Diabetes Mellitus, Type 1 ; Glucose ; Glycogen Synthase Kinase 3 beta/metabolism* ; Mice ; Phosphorylation ; Protein Kinase C/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; tau Proteins/metabolism*

OBJECTIVE: To identify whether naringenin plays a protective role during thoracic aneurysm formation in Marfan syndrome. METHODS: To validate the effect of naringenin, Fbn1^C1039G/+ mice, the mouse model of Marfan syndrome, were fed with naringenin, and the disease progress was evaluated. The molecular mechanism of naringenin was further investigated via in vitro studies, such as bioluminescence resonance energy transfer (BRET), atomic force microscope and radioligand receptor binding assay. RESULTS: Six-week-old Fbn1^C1039G/+ mice were fed with naringenin for 20 weeks. Compared with the control group, naringenin significantly suppressed the aortic expansion [Fbn1^C1039G/+ vs. Fbn1^C1039G/++naringenin: (2.49±0.47) mm, n=18 vs. (1.87±0.19) mm, n=22, P < 0.05], the degradation of elastin, and the expression and activity of matrix metalloproteinase 2 (MMP2) and MMP9 in the ascending aorta of Fbn1^C1039G/+ mice. Besides, treatment with naringenin for 6 weeks also attenuated the disease progress among the 20-week-old Fbn1^C1039G/+ mice with established thoracic aortic aneurysms [Fbn1^C1039G/+ vs. Fbn1^C1039G/++naringenin: (2.24±0.23) mm, n=8 vs. (1.90±0.17) mm, n=8, P < 0.05]. To understand the underlying molecular mechanisms, we examined the effects of naringenin on angiotensin Ⅱ type 1 receptor (AT1) signaling and transforming growth factor-β (TGF-β) signaling respectively, which were the dominant signaling pathways contributing to aortopathy in Marfan syndrome as previously reported. The results showed that naringenin decreased angiotensin Ⅱ (Ang Ⅱ)-induced phosphorylation of protein kinase C (PKC) and extracellular regulating kinase 1/2 (ERK1/2) in HEK293A cell overexpressing AT1 receptor. Moreover, naringenin inhibited Ang Ⅱ-induced calcium mobilization and uclear factor of activated T-cells (NFAT) signaling. The internalization of AT1 receptor and its binding to β-arrestin-2 with Ang Ⅱ induction were also suppressed by naringenin. As evidenced by atomic force microscope and radioligand receptor binding assay, naringenin inhibited Ang Ⅱ binding to AT1 receptor. In terms of TGF-β signaling, we found that feeding the mice with naringenin decreased the phosphorylation of Smad2 and ERK1/2 as well as the expression of TGF-β downstream genes. Besides, the serum level of TGF-β was also decreased by naringenin in the Fbn1^C1039G/+ mice. Furthermore, we detected the effect of naringenin on platelet, a rich source of TGF-β, both in vivo and in vitro. And we found that naringenin markedly decreased the TGF-β level by inhibiting the activation of platelet. CONCLUSION Our study showed that naringenin has a protective effect on thoracic aortic aneurysm formation in Marfan syndrome by suppressing both AT1 and TGF-β signaling.
Angiotensin II/metabolism* ; Animals ; Aortic Aneurysm, Thoracic/prevention & control* ; Calcium/metabolism* ; Disease Models, Animal ; Elastin/metabolism* ; Fibrillin-1/metabolism* ; Flavanones ; Marfan Syndrome/metabolism* ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; Mice ; Mice, Inbred C57BL ; Protein Kinase C/metabolism* ; Receptor, Angiotensin, Type 1/metabolism* ; Transforming Growth Factor beta/metabolism* ; Transforming Growth Factors/metabolism* ; beta-Arrestins/metabolism*

OBJECTIVE: To elucidate the underlying mechanism of Panax notoginseng saponin (PNS) on gastric epithelial cell injury and barrier dysfunction induced by dual antiplatelet (DA). METHODS: Human gastric mucosal epithelial cell (GES-1) was cultured and divided into 4 groups: a control, a DA, a PNS+DA and a LY294002+PNS+DA group. GES-1 apoptosis was detected by flow cytometry, cell permeability were detected using Transwell, level of prostaglandins E2 (PGE2), 6-keto-prostaglandin F1α (6-keto-PGF1α) and vascular endothelial growth factor (VEGF) in supernatant were measured by enzyme linked immunosorbent assay (ELISA), expression of phosphatidylinositide 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), Akt, phosphorylated-Akt (p-Akt), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), glycogen synthase kinase-3β (GSK-3β) and Ras homolog gene family member A (RhoA) were measured by Western-blot. RESULTS: DA induced apoptosis and hyper-permeability in GES-1, reduced supernatant level of PGE2, 6-keto-PGF1α and VEGF (P<0.05). Addition of PNS reduced the apoptosis of GES-1 caused by DA, restored the concentration of PGE2, 6-keto-PGF1α and VEGF (P<0.05). In addition, PNS attenuated the alteration of COX-1 and COX-2 expression induced by DA, up-regulated p-PI3K/p-Akt, down-regulated RhoA and GSK-3β. LY294002 mitigated the effects of PNS on cell apoptosis, cell permeability, VEGF concentration, and expression of RhoA and GSK-3β significantly. CONCLUSIONS PNS attenuates the suppression on COX/PG pathway from DA, alleviates DA-induced GES-1 apoptosis and barrier dysfunction through PI3K/Akt/ VEGF-GSK-3β-RhoA network pathway.
Cyclooxygenase 1 ; Epithelial Cells/metabolism* ; Glycogen Synthase Kinase 3 beta ; Humans ; Panax notoginseng ; Phosphatidylinositol 3-Kinases/metabolism* ; Platelet Aggregation Inhibitors ; Proto-Oncogene Proteins c-akt/metabolism* ; Saponins/pharmacology* ; Vascular Endothelial Growth Factor A ; rhoA GTP-Binding Protein

The aim of this study was to investigate the effect of Wnt5a on the vincristine (VCR) resistance in human ovarian carcinoma SKOV3 cells and its possible mechanism. The drug-resistant SKOV3/VCR cells were established by stepwise exposure to VCR, and then the SKOV3/VCR cells were stably transfected with specific shRNA interference plasmid vector targeting for Wnt5a. The mRNA expression level of Wnt5a was measured by RT-PCR. CCK-8 assay was used to detect the cell viability of SKOV3/VCR cells. The apoptosis was analyzed by flow cytometry. The protein expression levels of Wnt5a, MDR1, Survivin, β-catenin, Akt, p-Akt(S473), GSK3β and p-GSK3β(Ser9) were detected by Western blot. The result showed that SKOV3/VCR cells had significantly higher protein expression levels of Wnt5a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5a, compared with SKOV3 cells (P < 0.05). WNT5A gene silencing significantly increased the sensitivity of SKOV3/VCR cells to VCR, the IC of VCR being decreased from 38.412 to 9.283 mg/L (P < 0.05), synergistically enhanced VCR-induced apoptosis of SKOV3/VCR cells (P < 0.05), down-regulated the protein expression levels of MDR1, β-catenin and Survivin (P < 0.05), and inhibited phosphorylation of Akt and GSK3β (P < 0.05). Meanwhile, LY294002 (PI3K inhibitor) decreased the protein expression levels of MDR1, β-catenin and Survivin, as well as the phosphorylation levels of Akt and GSK3β in SKOV3/VCR cells (P < 0.05). These results suggest that WNT5A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.
ATP Binding Cassette Transporter, Subfamily B ; metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Gene Silencing ; Glycogen Synthase Kinase 3 beta ; metabolism ; Humans ; Ovarian Neoplasms ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Survivin ; metabolism ; Vincristine ; pharmacology ; Wnt-5a Protein ; metabolism

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.
Endothelial Cells* ; Gene Silencing ; Inflammation ; Intercellular Adhesion Molecule-1 ; Mitochondria ; Monocytes ; Phosphorylation ; Protein Kinase C beta* ; Protein Kinase C* ; Protein Kinases* ; Reactive Oxygen Species ; Superoxide Dismutase

OBJECTIVETo investigate the role of integrin-linked kinase (ILK) signaling pathway in the skin lesions and wound healing in diabetic rats.

METHODSThirty-six SD rats were divided into diabetic wound group (D) and non-diabetic wound group (N) according to the random number table, with 18 rats in each group. 10 g/L streptozocin (60 mg/kg) was intraperitoneally injected in rats in group D, while the rats in group N were given same quantity of sodium citrate buffer. Two weeks after successful reproduction of diabetic model of rats in group D, two full-thickness skin of an area of 2 cm × 2 cm was resected on both sides of back of rats in the two groups. Wounds of three rats of each group were photographed and examined on post injury day (PID) 1, 3, 7, 10, 14, and 21, and the wound healing rates were calculated. The non-injured skin and wound tissue (central part) on back of three rats of the rest 15 rats in the two groups were harvested on PID 3, 7, 10, 14, and 21, respectively. Morphology of the non-injured skin tissue was observed with HE staining, and the thickness of full-thickness skin and epidermis were measured. The mRNA expression levels of ILK, protein kinase B (Akt), and glycogen synthase kinase-3&beta; (GSK-3&beta;) in non-injured skin tissue were determined with real-time fluorescent quantitative RT-PCR. The protein expression levels of ILK, Akt, phosphorylated Akt, GSK-3&beta;, and phosphorylated GSK-3&beta; in non-injured skin tissue, and ILK, phosphorylated Akt in wound tissue were assessed with Western blotting. Data were processed with two independent-sample t test, one-way analysis of variance, SNK test and analysis of variance of factorial design.

RESULTS(1) After injury, the wound scabs of rats in group N were dry, and red granulation tissue with no excretion were seen when the scabs fell off, and the wound healed fast. After injury, excretion under the wound scabs of rats in group D was seen, and the scabs easily fell off with exposure of pink granulation tissue with much excretion, and the wounds healed slowly. Except for PID 3, the wound healing rate of rats in group D was significantly lower than that in group N on other PIDs (with t values from 3.858 to 13.738, P<0.05 or P<0.01). (2) On PID 3, the hair follicles and blood vessels in the non-injured skin tissue of rats in group N were rich, and the epidermis was composed of stratified cells in form of basal cells and keratinocyte, and the hair follicles and blood vessels in the non-injured skin tissue of rats in group D were scarce, and the epidermis was nearly composed of one-layer of cells. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N was similar from PID 3 to 21, and the thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group D on PID 3 was respectively (1 074 ± 66) and (15.1 ± 3.8) μm, and they gradually thinned out to (785 ± 122) and (9.7 ± 2.1) μm on PID 21, respectively. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N were significantly thicker than those in group D on each PID (with t values from 4.620 to 23.549, P values below 0.001). (3) From PID 3 to 21, the mRNA expression levels of ILK and Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values respectively 4.779 and 3.440, P values below 0.05), the mRNA expression levels of GSK-3&beta; in non-injured skin tissue of rats were similar in two groups (t=0.363, P>0.05). (4) From PID 3 to 21, the protein expression levels of ILK, Akt and phosphorylated Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values from 2.630 to 6.209, P<0.05 or P<0.01); the protein expression levels of GSK-3&beta; in non-injured skin tissue of rats in two groups were similar (t=0.652, P>0.05); the protein expression level of phosphorylated GSK-3&beta; in non-injured skin tissue of rats in group D was significantly higher than that in group N (t=4.131, P<0.001). The protein expression levels of ILK in wound tissue of rats in two groups were similar on each PID (with t values from 0.381 to 2.440, P values above 0.05). Except for PID 3, the protein expression levels of phosphorylated Akt in wound tissue of rats in group N were significantly higher than that in group D on other PIDs (with t values from 4.091 to 20.555, P<0.05 or P<0.01). From PID 3 to 21, the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group N were similar (F=2.522, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=117.329, P<0.001); the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group D were similar (F=1.337, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=184.120, P<0.001).

CONCLUSIONSThe skin lesion of diabetic rats may be related to the declined expression levels of ILK, Akt and phosphorylated Akt in the ILK signaling pathway. The refractory healing of wound in diabetic rats may be related to the declined expression level of phosphorylated Akt.

Animals ; Diabetes Mellitus, Experimental ; enzymology ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Phosphorylation ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Skin ; injuries ; Wound Healing

Hydrogen sulfide (HS) contributes to visceral hyperalgesia in primary sensory neurons, but its role in central nervous system remains largely unknown. This study was to investigate the roles and underlying mechanisms of HS and its endogenous synthesis enzymes in the arcuate nucleus (ARC) in rat pancreatic hyperalgesia. Chronic pancreatitis (CP) was induced in male adult Sprague-Dawley rats by intra-pancreatic ductal injection of trinitrobenzene sulfonic acid (TNBS). Abdominal hyperalgesia was assessed by referred somatic behaviors to mechanical stimulation of rat abdomen. Western blot analysis was performed to detect protein expression in the ARC. CP markedly upregulated cystathionine &beta;-synthetase (CBS) expression but did not alter cystathionine-γ-lyase level in the ARC at 4 weeks after TNBS injection. Although the expression of total GluN2B was not altered, CP greatly enhanced the phosphorylation level of GluN2B in the ARC when compared with age- and sex-matched control rats. CP also significantly increased expression of protein kinase Cγ (PKCγ) in the ARC. Arcuate microinjection of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA, an inhibitor of CBS) significantly attenuated abdominal pain in CP rats in a dose-dependent manner and reversed the CP-induced upregulation of p-GluN2B and PKCγ in the ARC. Furthermore, the GluN2B inhibitor or specific PKC inhibitor chelerythrine significantly attenuated abdominal hyperalgesia in CP rats. The p-GluN2B expression was also suppressed by PKC inhibitor. Taken together, our results suggest that the upregulation of CBS in the ARC leads to an activation of GluN2B via PKCγ, which may play an important role in generation of pain hypersensitivity of CP.
Acute Disease ; Animals ; Arcuate Nucleus of Hypothalamus ; Cystathionine beta-Synthase ; Hyperalgesia ; Male ; Pain ; Pancreatitis, Chronic ; Phosphorylation ; Protein Kinase C ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; Up-Regulation