DNMT1 involved in the analgesic effect of folic acid on gastric hypersensitivity through downregulating ASIC1 in adult offspring rats with prenatal maternal stress.

AIMS: Gastric hypersensitivity (GHS) is a characteristic pathogenesis of functional dyspepsia (FD). DNA methyltransferase 1 (DNMT1) and acid-sensing ion channel 1 (ASIC1) are associated with GHS induced by prenatal maternal stress (PMS). The aim of this study was to investigate the mechanism of DNMT1 mediating the analgesic effect of folic acid (FA) on PMS-induced GHS. METHODS: GHS was quantified by electromyogram recordings. The expression of DNMT1, DNMT3a, DNMT3b, and ASIC1 were detected by western blot, RT-PCR, and double-immunofluorescence. Neuronal excitability and proton-elicited currents of dorsal root ganglion (DRG) neurons were determined by whole-cell patch clamp recordings. RESULTS: The expression of DNMT1, but not DNMT3a or DNMT3b, was decreased in DRGs of PMS rats. FA alleviated PMS-induced GHS and hyperexcitability of DRG neurons. FA also increased DNMT1 and decreased ASIC1 expression and sensitivity. Intrathecal injection of DNMT1 inhibitor DC-517 attenuated the effect of FA on GHS alleviation and ASIC1 downregulation. Overexpression of DNMT1 with lentivirus not only rescued ASIC1 upregulation and hypersensitivity, but also alleviated GHS and hyperexcitability of DRG neurons induced by PMS. CONCLUSIONS: These results indicate that increased DNMT1 contributes to the analgesic effect of FA on PMS-induced GHS by reducing ASIC1 expression and sensitivity.

Overexpression of GRK6 alleviates chronic visceral hypersensitivity through downregulation of P2Y6 receptors in anterior cingulate cortex of rats with prenatal maternal stress.

AIMS: Visceral hypersensitivity is a major clinic symptom in patients with irritable bowel syndrome (IBS). Anterior cingulate cortex (ACC) is involved in processing the information of pain. Both G protein-coupled receptor kinase 6 (GRK6) and P2Y purinoceptor 6 (P2Y6) are associated with neuroinflammation and pathological pain. The aim of this study was to investigate the interaction between GRK6 and P2Y6 in ACC in the development of visceral hypersensitivity of adult offspring rats with prenatal maternal stress (PMS). METHODS: Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). The expression and cellular distribution of GRK6 and P2Y6 were determined by Western blotting, qPCR, and fluorescence immunohistochemistry. Co-immunoprecipitation was used to evaluate the interaction between GRK6 and P2Y6. RESULTS: The mRNA and protein levels of GRK6 were significantly decreased in ACC of PMS rats. The injection of GRK6 overexpression virus significantly attenuated visceral hypersensitivity of PMS rats. P2Y6's mRNA level, protein level, and ratio of membrane protein over total protein expression was markedly increased in PMS rats. P2Y6 antagonist MRS2578 microinjection reversed visceral hypersensitivity of PMS rats. GRK6 overexpression significantly reduced P2Y6's expression in membrane proteins and P2Y6's ratio of membrane protein over total protein expression. CONCLUSIONS: These results indicate that decreased GRK6 leads to the accumulation of P2Y6 at neuron membrane in ACC, thereby contributing to visceral hypersensitivity of PMS rats.

Upregulation of Spinal ASIC1 and NKCC1 Expression Contributes to Chronic Visceral Pain in Rats.

Aims: To determine whether acid-sensing ion channel 1 (ASIC1)-sodium-potassium-chloride cotransporter 1 (NKCC1) signaling pathway participates in chronic visceral pain of adult rats with neonatal maternal deprivation (NMD). Methods: Chronic visceral pain was detected by colorectal distension (CRD). Western blotting and Immunofluorescence were performed to detect the expression and location of ASIC1 and NKCC1. Whole-cell patch-clamp recordings were performed to record spinal synaptic transmission. Results: The excitatory synaptic transmission was enhanced and the inhibitory synaptic transmission was weakened in the spinal dorsal horn of NMD rats. ASIC1 and NKCC1 protein expression in the spinal dorsal horn was significantly up-regulated in NMD rats. Incubation of Amiloride reduced the amplitude of mEPSCs. Incubation of Bumetanide (BMT) increased the amplitude of mIPSCs. Intrathecal injection of ASIC1 or NKCC1 inhibitors reversed the threshold of CRD in NMD rats. Also, Amiloride treatment significantly reversed the expression of NKCC1 in the spinal dorsal horn of NMD rats. Conclusion: Our data suggest that the ASIC1-NKCC1 signaling pathway is involved in chronic visceral pain in NMD rats.