Signaling transduction of noxious visceral stimuli in postsynaptic dorsal column neurons in spinal cord

The spinal postsynaptic dorsal column pathway plays a critical role in the visceral pain transmission in spinal cord. The noxious visceral stimuli might induce complex receptor expression and intracellular signaling transductions in postsynaptic dorsal column neurons. It has been demonstrated that neurokinin-1(NK-1) receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and intracellular protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (MAPKs) and cAMP response element-binding (CREB) protein are involved in the signaling transduction of visceral stimuli. All these processes contribute to the sensitization of postsynaptic dorsal column neurons and enhance the spinal transmission of visceral pain, indicating a potential and promising way of visceral pain therapy to inhibit the sensitization of postsynaptic dorsal column neurons.     

Effects of oxidized high density lipoprotein on tissue factor expression in ECV304 cell line

AIM: To investigate the expression of tissue factor (TF) induced by oxidized high density lipoprotein (oxHDL) in human umbilical vein cell line, ECV304, and the related mechanisms. METHODS: Four main groups were designed: the negative, the positive (ECV304 with histamine), the HDL group and the oxHDL group. Quantitative real-time polymerase chain reaction (RQ-PCR) and Western blotting were used to detect the expression level of TF. The specific inhibitors of MAPKs, SP600125 (c-jun terminal NH2 kinase, JNK), SB203580 (p38 MAP kinase, p38 MAPK), PD98059 (extracellular signal-regulated kinase, ERK1/2) were used to investigate the underlying mechanisms. RESULTS: The TF expression in normal ECV304 cell line was not detected. Histamine administration resulted in a significant expression of TF in ECV304 cell line, with strongest effect after 1 h co-incubation at concentration of 1×10-5 mol/L histamine (about 4.8-fold higher expression of TF compared with that of 1×10-9 mol/L histamine). Expression level of TF was detected after stimulated with oxHDL in dose- and time- dependent manners. The highest expression of TF mRNA was found at 20 mg/L oxHDL and 6 h co-incubation, with 1.8-fold and 5.3-fold increase in TF expression, respectively, compared with that at 10 mg/L oxHDL and 2 h co-incubation. 20 mg/L oxHDL also caused an apparent augmentation of TF protein expression, about 1.5-fold higher compared with that stimulated by 40 mg/L oxHDL. HDL co-incubation did not cause a detectable expression of TF protein. The mRNA levels of TF in ECV304 cell line induced by oxHDL were decreased by 95.0%, 81.0%, 87.0%, respectively (all P<0.05) after application of inhibitors against p38MAPK, JNK and ERK1/2. CONCLUSION: The results suggest that oxHDL stimulates TF expression in ECV304 cell line in both dose- and time- dependent manners, in which MAPKs signal transduction may play an important role.     

Role of interleukin-1β in spinal LTP in rats with neuropathic pain

AIM: To investigate the role of interleukin-1β (IL-1β) in the long-term potentiation (LTP) of C-fiber-evoked field potentials in rats with neuropathic pain. METHODS: The rat model of neuropathic pain was produced by spared nerve injury (SNI) of sciatic nerve or the method of lumbar 5 ventral root transection (L5 VRT). The effect of exogenous IL-1β on C-fiber-evoked field potentials of spinal dorsal horn was tested in both intact rats and the rats with neuropathic pain. The roles of p38 MAPK and NF-κB in the process were also evaluated. RESULTS: IL-1β at concentration of 500 μg/L affected neither basal synaptic transmission mediated by C-fiber nor spinal LTP induced by high frequency stimulation in intact rats. However, low concentration (5 μg/L) of IL-1β induced LTP of C-fiber-evoked field potentials in the rats with neuropathic pain. Pretreatment with either p38 MAPK inhibitor (SB203580) or NF-κB inhibitor (PDTC) completely blocked LTP induced by IL-1β. CONCLUSION: Exogeneous IL-1β might induce spinal LTP of C-fiber-evoked field potentials in the rats with neuropathic pain. p38 MAPK and NF-κB may be involved in the process.     

Cucurbitin E relieves airway inflammation by inhibiting MAPKs/NF-κB signaling pathways in asthmatic mice

AIMTo investigate the effects of cucurbitacin E on airway inflammation and the signaling pathways of MAPKs and NF-κB in asthmatic mice. METHODSHealthy mice (n=40) were randomly divided into control group, model group, low-dose cucurbitacin E group, high-dose cucurbitacin E group and dexamethasone group. Ovalbumin sensitization was used to induce asthma in the mice. The protein levels of p-JNK, p-ERK1/2, p-p38 MAPK and p-p65 in the lung tissues were determined by Western blot. RESULTSCompared with control group, the numbers of inflammatory cells, such as eosinophils, lymphocytes and neutrophils, were significantly increased in model group, and the activity of MAPKs and NF-κB signaling pathway-related proteins was significantly enhanced. Cucurbitin E at high dose attenuated airway inflammation in asthmatic mice, and significantly inhibited the activity of MAPKs and NF-κB signaling pathway-related proteins. Histopathological results showed proliferation of goblet cells and bronchial mucosal epithelial cells, infiltration of inflammatory cells in the alveoli, and narrow alveolar cavity in model group, while the pathological changes were significantly alleviated in cucurbitin E treatment groups. CONCLUSION Cucurbitin E improves airway inflammation in asthmatic mice, and its mechanism may be related to the inhibition of MAPKs and NF-κB signaling pathways.     

Effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats

AIM:To investigate the effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats. METHODS:The male Sprague-Dawley rats were induced as the model of the type 2 diabetic neuropathic pain rats, they were randomly divided into 6 groups (n=27): type 2 diabetic neuropathic pain (DNP) group, type 2 diabetic neuropathic pain and intraperitoneal injection of curcumin (Cur) group, type 2 diabetic neuropathic pain and solvent control (DSC) group, type 2 diabetic neuropathic pain and JNK inhibitor (DJ) group, type 2 diabetic neuropathic pain and JNK inhibitor solvent control (DJS) group, type 2 diabetic neuropathic pain and monocyte chemoattractant protein 1 (MCP-1) agonist (DM) group. Another 27 normal SD rats were selected as control group. Mechanical withdrawal threshod and thermal withdrawal latency were measured at 3rd d, 7th d and 14th d after dosing, then the lumbar segment 4~6 of the spinal cord and L4~6 DRG were removed at the same time. ELISA was used to measure MCP-1 level. The expression of p-JNK was determined by Western blotting. RESULTS:Compared with DNP group, p-JNK was significantly decreased at 7th d and 14th d in Cur group, DJ group and DM group after treatment (P<0.05). Compared with C group, the MCP-1 was significantly declined in other 6 group after streptozotocin injection (P<0.05). Compared with DNP group, MCP-1 were significantly increased at 7th d and 14th d in Cur group and DJ group after treatment (P<0.05), and that in DM group was greatly decreased (P<0.05). CONCLUSION: The expression of p-JNK and MCP-1 was increased in DNP rats with spinal cord and dorsal root ganglion. The mechanism of curcumin reducing the neuropathic pain in type 2 diabetic rats might be through regulating the JNK/MCP-1 pathway.     

Role of intestinal myofibroblasts in IBD-associated intestinal fibrosis

Intestinal fibrosis is the complications of inflammatory bowel disease (IBD), intestinal myofibroblast cells are the key to intestinal fibrosis. Intestinal myofibroblasts and its interaction with inflammatory cells play an important role in IBD-related intestinal fibrosis.     

LPS induced B7-H1 expression in pancreatic carcinoma Panc-1 cells

AIM: To explore the mechanism of lipopolysaccharide (LPS)-induced B7-H1 expression in pancreatic carcinoma cell line Panc-1. METHODS: The levels of phosphorylated p38 mitogen-activated protein kinase (p-p38), phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated c-Jun N-terminal kinase (p-JNK) after stimulated with LPS or treated with mitogen-activated protein kinases (MAPKs) inhibitors were detected by Western blotting. The expression of B7-H1 in Panc-1 cells after LPS stimulation or MAPKs inhibitor treatment was measured by real-time PCR and Western blotting. RESULTS: The levels of B7-H1, p-p38, p-ERK and p-JNK were up-regulated with LPS stimulation. The promoted p-p38, p-ERK and p-JNK levels induced by LPS were inhibited by the corresponding MAPKs inhibitors. Furthermore, the inhibitors of p38 and ERK attenuated LPS-induced B7-H1 expression. However, JNK inhibitor had very little effect on LPS-induced B7-H1 expression. CONCLUSION: LPS induces B7-H1 expression in pancreatic carcinoma cell line Panc-1. ERK and p38 are involved in this regulation as the inhibitors of ERK and p38 attenuate LPS-induced B7-H1 expression.     

Effect of endoplasmic reticulum stress protein BiP on type 2 diabetic neuropathic pain in rats treated with curcumin

AIM: To investigate the effects of immunoglobulin heavy chain-binding protein (BiP),an endoplasmic reticulum stress protein, on mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), spinal dorsal horn and dorsal root ganglion (DRG) in type Ⅱ diabetic neuropathic pain rats treated with curcumin. METHODS: The rats were fed with a high-fat and high-fructose diet for 8 weeks to induce insulin resistance, and then were intraperitoneally injected with streptozotocin (STZ, 35 mg/kg). Eighty-one rats were selected into experimental design as their blood glucose ≥ 16.7 mmol/L 3 d after STZ injection and their MWT and TWL were decreased to 85% of the baseline values 14 d after STZ injection. The rats were divided into 3 groups (n=27 each): DNP group: type 2 diabetic neuropathic pain; DCur group: type 2 diabetic neuropathic pain and intraperitonal injection of curcumin at a dose of 100 mg·kg^-1·d^-1; DSC group: type 2 diabetic neuropathic pain and intraperitonal injection of corn oil at a dose of 4 mL/kg. Another 27 normal SD male rats fed with normal forage were adopted as control group (C group). MWT and TWL were measured at the time points of 3 d, 7 d and 14 d after curcumin injection. The lumbar segment 4~6 of the spinal cord and the corresponding DRG were removed at the same time. The expression of BiP was determined by immunohistochemical staining and Western blotting. RESULTS: Compared with C group, the rats in DNP group developed hyperglycemia and a decrease in MWT and TWL, as well as an increase in the activity of BiP in spinal dorsal horn and DRG (P<0.05). Compared with DNP group, the rats in DCur group at the time point of 7 d significantly attenuated mechanical allodynia and thermal hyperalgesia, and these effects were correlated with the inhibition of BiP hyper-activation at the time point of 14 d after treatment with curcumin (P<0.05). No significant difference of MWT, TWL and the expression of BiP between DNP group and SC group was observed. CONCLUSION: BiP participates in the pathogenesis of type Ⅱ diabetic neuropathic pain. Curcumin attenuates the MWT and TWL in type 2 diabetic neuropathic pain rats. The mechanism may be involved in the inhibition of BiP expression by curcumin.     

Signal transduction in cells induced by endotoxin

Depending on LBP/CD14 systems, LPS activates a series of signal-transducing systems in cells. Protein tyrosin kinase (PTK) system, ceramide activated kinase (CAK) system might play an important role in cells signal-transducing. This article give a summary about signal transduction in cells induced by endotoxin.     

The role of Src-family kinases in long-term potentiation in rat spinal dorsal horn

AIM: To investigate the role of Src-family kinases (SFKs) in long-term potentiation (LTP) of C-fiber-evoked potentials in rat spinal dorsal horn and the underlying mechanism. METHODS: The effect of SFKs inhibitors on spinal LTP induced by high-frequency stimulation of sciatic nerve was determined by electrophysiological method. The levels of phosphorylated SFKs (p-SFKs) in spinal dorsal horn were measured by Western blotting at different time points after LTP induction. The cell types that expressed p-SFKs following LTP induction were determined by double-labeled immunofluorescence staining. RESULTS: Electrophysiological data revealed that pretreatment with SFKs inhibitors (PP2 or SU6656) enabled high-frequency stimulation to induce long-term depression (LTD) rather than LTP in spinal dorsal horn. Western blotting analysis showed that the level of p-SFKs in ipsilateral spinal dorsal horn increased at 15 min after LTP induction. Double-labeled immunofluorescence staining demonstrated that p-SFKs were highly restricted to spinal microglia. CONCLUSION: SFKs in microglia play a critical role in the induction of LTP in spinal dorsal horn. Inhibition of SFKs and the downstream molecules may be helpful for curing neuropathic pain.     

Analgesic effect of angiotensin angiotensin Ⅱ type 2 receptor antagonist EMA401 on neuropathic pain in rats and its mechanism

AIM: To explore whether angiotensin Ⅱ type 2 receptor antagonist EMA401 decreases neuropathic pain and the expression of growth-associated protein-43 (GAP-43), protein kinase C (PKC) and calmodulin (CaM) in dorsal root ganglia (DRG) during chronic constriction injury (CCI) in rats. METHODS: SD rats were used to establish CCI model and randomly divided into 4 groups. The rats in model group were given equal volume of normal saline by intragastric administration. The rats in low dose (LD) group were given 5 mg/kg EMA401 by intragastric administration. The rats in middle dose (MD) group were given 10 mg/kg EMA401 by intragastric administration. The rats high dose (HD) group were given 20 mg/kg EMA401 by intragastric administration. The rats in sham operation group received equal volume of normal saline by intragastric administration. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were measured before operation and 7 d, 14 d and 28 d after CCI. After behavioral test, DRG of lumbar spinal was obtained from each group, and was used to determine Ca²⁺ concentration by o-cresolphthalein complexone microplating method, and the expression of GAP-43, PKC and CaM at mRNA and protein levels by Western blotting and RT-PCR. RESULTS: Compared with model group, EMA401 significantly increased the TWL and MWT (P<0.05). Meanwhile, EMA401 significantly reduced Ca²⁺ concentration and the expression of GAP-43, PKC and CaM at mRNA and protein levels in the DRG (P<0.05). CONCLUSION: EMA401 may attenuate neuropathic pain of CCI by inhibiting Ca²⁺ concentration and the expression of GAP-43, PKC and CaM.     

Effect of Shaofu-Zhuyu decoction on MAPK/ERK signaling pathway in rats with endometriosis

AIM: To observe the effects of Shaofu-Zhuyu decoction (SFZY) on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway in the rats with endometriosis (EM), and to explore the mechanism of SFZY for treatment of EM.METHODS: Healthy female SD rats were used to establish the EM model. The rats were randomly divided into blank control group, model group, positive control group, and low dose, middle dose and high dose of SFZY groups. The pathological changes of the endometriotic tissue were observed by HE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and IL-8 in the uterine tissue were detected by ELISA. The mRNA expression of ERK, vascular endothelial growh factor (VEGF) and matrix metalloprotein-9 (MMP-9) was detected by RT-qPCR. The protein expression of nuclear factor-κB (NF-κB), MAPK and MAPK-ERK kinase (MEK) was determined by Western blot.RESULTS: Compared with model group, the levels of TNF-α, IL-6 and IL-8 in the uterine tissue of the rats in middle dose and high dose of SFZY groups were significantly decreased (P<0.05), the mRNA expression of ERK, VEGF and MMP-9 was significantly reduced, and the protein expression of NF-κB, MEK and MAPK was decreased significantly in the rat endometriotic tissues (P<0.05).CONCLUSION: SFZY may play a key role in the treatment of EM by regulating MAPK/ERK signaling pathway.     

Resveratrol attenuates inflammatory pain induced by complete Freund's adjuvant via NF-κB signaling pathway in a mouse model

AIM: To investigate the anti-inflammatory action of resveratrol (Res) and its correlation with nuclear factor-κB (NF-κB) signaling pathway in a mouse model of inflammatory pain.METHODS: BALB/c mice (n=60) were randomly divided into 6 groups:normal control group, inflammatory pain model group, positive control (dexamethasone, 0.5 mg/kg) group and resveratrol (100, 50 and 25 mg/kg) groups (10 mice in each group). In order to observe the anti-inflammatory pain effects of reseratrol on mice, the paw withdrawal mechanical threshold, paw withdrawal thermal latency and cold withdrawal times were detected. In order to analyze the mechanism of analgesic effect of resveratrol, the expression levels of NF-κB, inhibitor of NF-κB (IκB) α, inhibitor of NF-κB kinase (IKK) β, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the spinal cord tissues (L4~L6) of the mice were determined by RT-PCR and Western blot.RESULTS: The resveratrol at 100 and 50 mg/kg increased the paw withdrawal mechanical threshold, prolonged the paw withdrawal thermal latency, and decreased the cold withdrawal times in the inflammatory pain mice (P<0.05 or P<0.01). The resveratrol at 100 mg/kg down-regulated the mRNA and protein expression levels of NF-κB, IκBα, IKKβ, TNF-α and IL-1β in the spinal cord tissues (L4~L6) of inflammatory pain mice (P<0.05 or P<0.01).CONCLUSION: Resveratrol ameliorates the inflammatory pain of the mice induced by complete Freund's adjuvant. The mechanism is related to the inhibition of NF-κB signaling pathway.     

Changes of subfamilies of mitogen-activated protein kinases in recovery process of acute renal ischemia/reperfusion injury

AIM: To investigate the change and the possible role of mitogen-activated protein kinase(MAPK) subfamilies in early recovery process following acute renal ischemia/reperfusion injury. METHODS: Ischemia/reperfusion renal injury model was made by placing an atraumatic vascular clamp in renal pedicel. The morphologic change was observed by transmission electron microscope. The proliferating cell nuclear antigen(PCNA) positive renal cells were detected by immunohistochemistry. Extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase(JNK) activity was assayed by specific substrate phosphorylation with immunoprecipitation. RESULTS: After acute ischemia, microvilli in renal tubular cells appeared again after 2h of reperfusion. At the same time, the PCNA-positive cells were initially increased. ERK activity decreased at 45 min of ischemia, and completely recovered at 5 min of reperfusion. JNK activity was not influenced by ischemia, but increased at 5 min of reperfusion, reaching its maximal activity at 20 min of reperfusion, and prolonged within 2h reperfusion. CONCLUSION: After renal ischemia/reperfusion injury, the early recovery of renal tubule damage was related to the changes of MAPKs in which the increase of JNK activity might be more important.     

Effect of HO-1 expression on neuronal apoptosis in spinal cord of rats with formaldehyde inflammatory pain

AIM: To observe the change of heme oxygenase(HO-1)expression in the spinal cord during formaldehyde-induced pathological pain and investigate the effect of HO-1 on neuronal apoptosis in the spinal cord induced by formaldehyde inflammatory pain.METHODS: The protein expression of HO-1 in the left and the right spinal dorsal horn was detected by Western blotting. The neuronal apoptosis rate of the spinal cord was determined by flow cytometry.RESULTS: Compared to control group, the protein expression of HO-1 was significantly up-regulated at different time points after the injection of formaldehyde, which was most obviously 24 h after the injection of formaldehyde and was still higher than that in control group at 72 h. Compared to control group, the neuronal apoptosis rate of spinal cord increased in the rats with formaldehyde inflammatory pain. No significant difference of the neuronal apoptosis rate was observed between formaldehyde group and solvent control group . Intrathecal injection of 100 μg ZnppIX, an inhibitor of HO-1, attenuated the degree of spontaneous pain response, but induced an increase in the rate of neuronal apoptosis in spinal cord in the rats with formaldehyde inflammatory pain. CONCLUSION: Formaldehyde inflammatory pain induces the increases in HO-1 expression and neuronal apoptosis in the rat spinal cord. HO-1 promotes the response of spontaneous pain and inhibits the process of neuronal apoptosis in spinal cord induced by formaldehyde inflammatory pain.     

Effects of protein kinase C inhibitor,chelerythrine chloride,on nitric oxide synthase expression and nitric oxide content in spinal cord of rats with inflammatory pain induced by formalin

AIM: To study the effects of protein kinase C (PKC) inhibitor， chelerythrine chloride (CH)， on nociceptive response, nitric oxide synthase (NOS) expression and nitric oxide (NO) content in spinal cord of rats with inflammatory pain. METHODS: Inflammatory pain was induced by formalin injection into right hind paw. NADPH-d histochemistry was used to investigate the changes of NOS expression. Nitrate/nitrite (NO2-/NO3-) was assayed to represent NO content. RESULTS: Compared with the control group, the number of NADPH-d positive cells increased significantly in the superficial layer (LaminaeⅠ-Ⅱ) of the spinal cord dorsal horn and the grey matter surrounding the central canal (Laminae Ⅹ) in rats with inflammatory pain, the reactive degree of NADPH-d positive soma and fibers and NO content of the lumbar enlargement of spinal cord also increased significantly. Intrathecal injection of CH inhibited the spontaneous pain response in the second phase induced by formalin injection, and prevented the increases in the number and reactive degree of NADPH-d positive cells, as well as NO content of the lumbar enlargement of spinal cord. CONCLUSION: It is suggested that the activation of PKC promotes NOS expression and NO production in the nociceptive neurons of spinal cord during formalin-induced inflammatory pain.     

Effects of genistein on NF-κB in aortas of ovariectomized rats

AIM: To investigate the effect of genistein on the expression of NF-κB p65 in aortas of ovariectomized rats. METHODS: Forty female Wistar rats were randomly divided into four groups: sham-operated (control), ovariectomized (OVX), ovariectomized with 17 β-estradiol replacement (OVX+E2), ovariectomized with genistein replacement (OVX+GST) group. Six weeks after treatment, the rats were killed and the expression of NF-κB p65 in the aorta was observed by immunohistochemistry. RESULTS: The expression of NF-κB p65 was much more in OVX aortas than that in control aortas (P<0.05), while being much less in OVX+E2 and OVX+GST aortas. Pathological changes were found in aorta of the ovariectomized rats. CONCLUSION: Genistein inhibits the expression of NF-κB p65 in aortas of ovariectomized rats and may play an important role in the pathogenesis of atherosclerosis.