TET1 Participates in Complete Freund's Adjuvant-induced Trigeminal Inflammatory Pain by Regulating Kv7.2 in a Mouse Model.

Trigeminal inflammatory pain is one of the most severe pain-related disorders in humans; however, the underlying mechanisms remain largely unknown. In this study, we investigated the possible contribution of interaction between ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and the voltage-gated K+ channel Kv7.2 (encoded by Kcnq2) to orofacial inflammatory pain in mice. We found that complete Freund's adjuvant (CFA) injection reduced the expression of Kcnq2/Kv7.2 in the trigeminal ganglion (TG) and induced orofacial inflammatory pain. The involvement of Kv7.2 in CFA-induced orofacial pain was further confirmed by Kv7.2 knockdown or overexpression. Moreover, TET1 knockdown in Tet1flox/flox mice significantly reduced the expression of Kv7.2 and M currents in the TG and led to pain-like behaviors. Conversely, TET1 overexpression by lentivirus rescued the CFA-induced decreases of Kcnq2 and M currents and alleviated mechanical allodynia. Our data suggest that TET1 is implicated in CFA-induced trigeminal inflammatory pain by positively regulating Kv7.2 in TG neurons.

Spinal Sirtuin 3 Contributes to Electroacupuncture Analgesia in Mice With Chronic Constriction Injury-Induced Neuropathic Pain.

BACKGROUND: Electroacupuncture (EA) is a traditional Chinese therapeutic technique that has a beneficial effect on neuropathic pain; however, the specific mechanism remains unclear. In this study, we investigated whether EA inhibits spinal Ca/calmodulin-dependent protein kinase II (CaMKIIα) phosphorylation through Sirtuin 3 (SIRT3) protein, thus relieving neuropathic pain. MATERIALS AND METHODS: We used wild-type and SIRT3 knockout (SIRT3-/-) mice and used chronic constriction injury (CCI) as a pain model. We performed Western blotting, immunostaining, von Frey, and Hargreaves tests to gather biochemical and behavioral data. Downregulation and overexpression and spinal SIRT3 protein were achieved by intraspinal injection of SIRT3 small interfering RNA and intraspinal injection of lentivirus-SIRT3. To test the hypothesis that CaMKIIα signaling was involved in the analgesic effects of EA, we expressed CaMKIIα-specific designer receptors exclusively activated by designer drugs (DREADDs) in the spinal dorsal horn (SDH) of mice. RESULTS: These results showed that the mechanical and thermal hyperalgesia induced by CCI was related to the decreased spinal SIRT3 expression in the SDH of mice. A significant reduction of mechanical and thermal thresholds was found in the SIRT3-/- mice. SIRT3 overexpression or EA treatment alleviated CCI-induced neuropathic pain and prevented the spinal CaMKIIα phosphorylation. Most importantly, EA increased the expression of spinal SIRT3 protein in the SDH. Downregulation of spinal SIRT3 or CaMKIIα Gq-DREADD activation inhibited the regulatory effect of EA on neuropathic pain. CONCLUSION: Our results showed that CaMKIIα phosphorylation was inhibited by spinal SIRT3 in neuropathic pain and that EA attenuated CCI-induced neuropathic pain mainly by upregulating spinal SIRT3 expression in the SDH of mice.

Role of spinal RIP3 in inflammatory pain and electroacupuncture-mediated analgesic effect in mice.

AIMS: Electroacupuncture (EA) is a potentially useful treatment for inflammatory pain. Receptor-interacting protein 3 (RIP3) triggers the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome; activation independent of necroptosis has been reported. However, the role of RIP3 in inflammatory pain and its EA-induced analgesic effects remains unclear. MAIN METHODS: Mice were treated with EA (2 Hz, 2 mA) after complete Freund's adjuvant (CFA) pain models were established. Inhibition or activation of spinal RIP3 was achieved by intrathecal administration of GSK-843 (a specific RIP3 inhibitor) or microinjection of lentivirus-RIP3, respectively. Mechanical analgesiometry and thermal analgesiometry were used to assess paw withdrawal threshold and paw withdrawal latency in mice. Quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to evaluate the expression of RIP3 and NLPR3 in spinal dorsal horn (SDH) of mice. KEY FINDINGS: The expression of spinal RIP3 and NLPR3 increased significantly after CFA injection. Both intrathecal administration of GSK-843 and EA alleviated mechanical and thermal pain behaviors induced by CFA and inhibited the expression of RIP3 and NLRP3 in the SDH of CFA mice. Over-expression of RIP3 induces pain-like symptoms in mice and inhibits the regulatory effects of EA on inflammatory pain. SIGNIFICANCE: Our results indicate that the EA analgesia effect may be related to suppression of RIP3 and NLRP3 expression in the SDH. This study could provide potential insights into the underlying spinal mechanisms involved in the analgesic effect of EA.

Glucocorticoid Receptor Contributes to Electroacupuncture-Induced Analgesia by Inhibiting Nav1.7 Expression in Rats With Inflammatory Pain Induced by Complete Freund's Adjuvant.

BACKGROUND: While electroacupuncture (EA) has been used traditionally for the treatment of chronic pain, its analgesic mechanisms have not been fully clarified. We observed in an earlier study that EA could reverse inflammatory pain and suppress high Nav1.7 expression. However, the molecular mechanism underlying Nav1.7 expression regulation is unclear. In this study, we studied the relationship between the glucocorticoid receptor (GR) and Nav1.7 and the role of these molecules in EA analgesia. MATERIALS AND METHODS: In this study, we established an inflammatory pain model by intraplantar injection of complete Freund's adjuvant (CFA) in rats. EA stimulation was applied to the ipsilateral "Huantiao" (GB30) and "Zusanli" (ST36) acupoints in the rat model. Western blotting, real-time polymerase chain reaction, immunostaining, intrathecal injection, and chromatin immunoprecipitation (ChIP) assay were performed to determine whether the sodium channel protein Nav1.7 plays a role in CFA-induced pain and whether GR regulates Nav1.7 expression during analgesia following EA stimulation. RESULTS: EA application significantly decreased the paw withdrawal threshold thresholds and thermal paw withdrawal latency and suppressed GR and Nav1.7 expression in the dorsal root ganglion. Moreover, treatment with a GR sense oligonucleotide (OND) markedly reversed these alterations. In contrast, treatment with a GR antisense OND along with EA application exerted a better analgesic effect, which was accompanied by the suppression of Nav1.7 and GR protein expression. The ChIP assay showed that the binding activity of GR to the Nav1.7 promoter was enhanced in CFA injected rats and suppressed in EA-treated rats. CONCLUSIONS: The present study demonstrated that EA exerted anti-hyperalgesic effects by inhibiting GR expression, which led to Nav1.7 expression modulation in the rat model of CFA-induced inflammatory pain.

[Mechanism of Calculus Bovis Sativus in inhibiting hepatocyte lipid deposition based on serum pharmacology].

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.

Investigation of the synergistic effects of haloperidol combined with Calculus Bovis Sativus in treating MK-801-induced schizophrenia in rats.

Clinical studies that focused on treating schizophrenia showed that Calculus Bovis Sativus (CBS), a substitute of Calculus Bovis, when used in combination with haloperidol could significantly lower the dosage of haloperidol compared with treatment with haloperidol alone, whereas efficacy was maintained. The aim of this study was to investigate the synergetic anti-schizophrenia effects in rats using CBS in combination with haloperidol. An open field test was conducted to verify the pharmacodynamic effects of a combination treatment of CBS and haloperidol on MK-801-induced schizophrenic rats. Rat plasma concentrations of intragastric haloperidol and intravenous haloperidol were determined after oral administration of a single dose or 1-week of pretreatment with CBS (50 mg/kg). The pharmacodynamic data showed a significant decrease in locomotor activity and an increase in the percentage of the central distance when haloperidol was concomitantly administered with CBS compared with haloperidol administration alone. The AUC0-∞ and Cmax of haloperidol in the orally coadministered groups were significantly higher compared with the oral treatment with haloperidol alone. In conclusion, oral coadministration of CBS with haloperidol resulted in a synergistic effect in rats. The enhanced oral bioavailability of haloperidol when combined with CBS might be attributed to the interaction between them.

Neuroprotective effect of three caffeic acid derivatives via ameliorate oxidative stress and enhance PKA/CREB signaling pathway.

This study was conducted to elucidate the neuroprotective effect of caffeic acid phenethyl ester (CAPE), (R)-2-Hydroxy-3-(3,4-dihydroxyphenyl) propionic acid (Danshensu) and Curcumin, three caffeic acid derivatives which are contained in fruits, grains and certain dietary supplements. Our results showed that these compounds significantly attenuated H2O2-induced toxicity in PC12 cells in a dose-dependent manner. Furthermore, these compounds significantly improved the behavioral performance of d-gal-treated mice in both step-down avoidance test and Morris water maze test. Biochemical examination and western blot analysis showed that these compounds could ameliorate oxidative stress and facilitate activation of the protein kinase A (PKA)-cyclic AMP response element-binding protein (CREB) pathway. Its beneficial effects may partly relate to enhancing the activity of endogenous antioxidant enzymes and modulating the PKA/CREB signaling pathway. Furthermore, our results also indicated that the presence of 3, 4-dihydroxyphenyl groups in A ring may enhance their neuroprotective activity.