Chunping Zhang

LncRNA NONRATT021972 siRNA regulates neuropathic pain behaviors in type 2 diabetic rats through the P2X 7 receptor in dorsal root ganglia

BackgroundLong non-protein-coding RNAs (lncRNAs) are involved in the pathological processes of nervous system diseases. NONRATT021972 is an lncRNA. This study explores the effects of lncRNA NONRATT021972 small interference RNA (siRNA) on diabetic neuropathic pain (DNP) mediated by the P2X7 receptor in the rat dorsal root ganglia (DRG).ResultsOur results show that NONRATT021972 expression was significantly higher in the DRG of diabetes mellitus (DM) group compared with control group. NONRATT021972 expression in the DRG was reduced when DM rats were treated with NONRATT021972 siRNA. NONRATT021972 siRNA treatment in type 2 DM rats increased the mechanical withdrawal threshold (MWT), the thermal withdrawal latency (TWL) and the sensory nerve conduction velocity (SNCV) of rat tail nerves. After intravenous injection with NONRATT021972 siRNA in DM rats, the P2X7, GFAP and TNF-ɑ expression levels in DRG were decreased. An interaction between the RNA (NONRATT021972) and protein (P2X7) was predicted by the application of bioinformatics technology. The BzATP-activated currents in DRG non-neurons (satellite glial cells) of DM rats were significantly increased compared to control rats. NONRATT021972 siRNA treatment inhibited the ATP-activated currents in HEK293 cells transfected with pEGFP-P2X7.ConclusionsNONRATT021972 siRNA treatment can decrease the expression levels of P2X7 mRNA and protein and inhibit the activation of satellite glial cells (SGCs) in the DRG of type 2 DM rats. Moreover, NONRATT021972 siRNA treatment reduced the release of inflammatory factors (TNF-α), thereby inhibiting the excitability of DRG neurons and reducing mechanical and thermal hyperalgesia in type 2 DM rats.

Role of P2X3 receptor in myocardial ischemia injury and nociceptive sensory transmission

Extracellular ATP acts on purinergic receptors as a potent agonist for a variety of different cell types, including cardiomyocytes and nodose ganglia. P2X3 receptor is the most abundant P2X-receptor subtype in heart and nodose ganglia. This study wants to observe the role of P2X3 receptor in myocardial ischemic injury and nociceptive transmission via nodose ganglia. The serum lactate dehydrogenase (LDH), creatine kinase (CK) and CK isoform MB (CK-MB) activities were measured by automatic biochemistry analyzer. The electrocardiogram (ECG) recorded ST-segment changes and cardiac arrhythmia. The expression of P2X3 immunoreactivity, mRNA and protein were analyzed by immunohistochemistry, in situ hybridization and western blotting. Myocardial ischemia enhanced the serum LDH, CK and CK-MB activities and caused premature beats. P2X3 receptor antagonist A-317491 decreased the serum enzyme activities and improved premature beats in myocardial ischemic rats. The expression of P2X3 mRNA and protein in the ischemic injury heart were higher than that in the naive heart as control. A-317491 reduced the expression of P2X3 mRNA and protein in the myocardial ischemic injury. The myocardial ischemic injury increased the expression of P2X3 immunoreactivity and mRNA in nodose ganglia. In rats treated with A-317491, the expression of P2X3 immunoreactivity and mRNA in nodose ganglia was reduced. Blocking the nociceptive transmission mediated by P2X3 receptor may protect the cardiac function. According to these results, P2X3 receptor could be thought of as a new target for treating myocardial ischemic injury and cardiac arrhythmia and inhibiting nociceptive transmission of myocardial ischemic injury.

LncRNA uc.48+ is involved in diabetic neuropathic pain mediated by the P2X3 receptor in the dorsal root ganglia

Some long non-coding RNAs (lncRNAs) participate in physiological processes that maintain cellular and tissue homeostasis, and thus, the dysregulated expression of lncRNAs is involved in the onset and progression of many pathological conditions. Research has indicated that the genetic knockout of some lncRNAs in mice resulted in peri- or postnatal lethality or developmental defects. Diabetes mellitus (DM) is a major cause of peripheral neuropathy. Our studies showed that the expression levels of lncRNA uc.48+ in the diabetic rat dorsal root ganglia (DRG) and the DM patients’ serum samples were increased. It suggested that lncRNA uc.48+ was involved in the pathophysiological process of DM. The aim of this study was to investigate the effects of lncRNA uc.48+ small interfering RNA (siRNA) on diabetic neuropathic pain (DNP) mediated by the P2X3 receptor in the DRG. The values of the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured by the von Frey test and Hargreaves’ test, respectively. The levels of P2X3 protein and messenger RNA (mRNA) in the DRG were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and western blotting. The experiments showed that the MWT and TWL values in DM rats were lower than those in the control rats. The MWT and TWL values in DM rats treated with lncRNA uc.48+ siRNA were increased compared to those in DM rats, but there was no significant difference between the DM rat group and the DM + scramble siRNA group. The levels of P2X3 protein and mRNA in the DM DRG were higher than those in the control, while the levels of P2X3 protein and mRNA in the DG of DM rats treated with uc.48+ siRNA were significantly decreased compared to those in DM rats. The expression levels of TNF-α in the DRG of DM rats treated with uc.48+ siRNA were significantly decreased compared to those in the DM group. The phosphorylation and activation of ERK1/2 in the DM DRG were decreased by uc.48+ siRNA treatment. Therefore, uc.48+ siRNA treatment may alleviate the DNP by inhibiting the excitatory transmission mediated by the P2X3 receptor in DRG.

Puerarin blocks the signaling transmission mediated by P2X3 in SG and DRG to relieve myocardial ischemic damage.

P2X₃ receptors in stellate ganglia (SG) and cervical dorsal root ganglia (DRG) neurons are involved in sympathoexcitatory reflex induced by myocardial ischemic damage. Puerarin, a major active ingredient extracted from the traditional Chinese plant medicine Ge-gen, has been widely used in treatment of myocardial and cerebral ischemia. The present study is aimed to observe the effects of puerarin on the signaling transmission mediated by P2X₃ receptor in SG and DRG after myocardial ischemic damage. Our results showed that systolic blood pressure and heart rate increased, and the expression levels of P2X₃ mRNA and protein in SG and DRG were up-regulated after myocardial ischemic damage. Puerarin reduced systolic blood pressure and heart rate, relieved pain and decreased up-regulated expression of P2X₃ mRNA and protein in SG and DRG after myocardial ischemia. Puerarin inhibited the up-regulated ATP-activated currents in DRG neurons after myocardial ischemia. Thus, puerarin can relieve myocardial ischemic damage through blocking the P2X₃ signaling transmission and then depressed the aggravated sympathoexcitatory reflex.

The involvement of P2X3 receptors of rat sympathetic ganglia in cardiac nociceptive transmission

In this work we have examined the effects of P2X3 receptor antagonist A-317491 on P2X3 expression in superior cervical ganglion (SCG) from naive and myocardial ischemic rats to observe the effect of P2X3 receptors in cardiac nociceptive transmission. A-317491 improved nociceptive behavior. In the ganglia neurons of rats at 14 days after myocardial ischemic injury, the staining of P2X3 receptor in myocardial ischemic groups appeared to be more intense than those of naive rats detected by immunohistochemistry. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 immunoreactivity was lower than that in myocardial ischemic rats. The signals of P2X3 and its protein and mRNA in myocardial ischemic groups were higher than those in control group measured by western blotting and in situ hybridization. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 and its mRNA was lower than that in myocardial ischemic rats. These results suggest the involvement of P2X3 receptors in cardiac nociceptive transmission and A-317491 may inhibit the transmission mediated by P2X3 receptors in rat SCG after myocardial ischemia.ResumenCon el fin de observar el efecto de los receptores P2X3 en la transmisión nociceptiva cardiaca, se estudia la influencia de A-317491, antagonista de los receptores P2X3, sobre su expresión de en le ganglio cervical superior, tanto en ratas control como en las sometidas a isquemia miocárdica. En ratas de 14 días, se comprueba mediante inmunohistoquímica que las neuronas de los ganglios de las sometidas a isquemia miocárdica presentan mayor intensidad de tinción de P2X3 que las ratas control, lo que se atenúa con el tratamiento con A-317491. La expresión de P2X3 y del mRNA correspondiente en las ratas isquémicas es superior a las ratas control y el tratamiento con el antagonista reduce el incremento. Lo resultados sugieren la implicación de los receptores P2X3 en la transmisión nociceptiva cardiaca y que el antagonista A-317491 puede inhibir la transmisión mediada por P2X3 en el ganglio superior cervical de la rata.

Erratum to: The effect of sinomenine in diabetic neuropathic pain mediated by the P2X3 receptor in dorsal root ganglia

Type 2 diabetes mellitus (T2DM) accounts for more than 90% of all cases of diabetes mellitus (DM). Diabetic neuropathic pain (DNP) is a common complication of T2DM. Sinomenine is a natural bioactive component extracted from the Sinomenium acutum and has anti-inflammatory effects. The aim of our study was to investigate the effects of sinomenine on DNP mediated by the P2X3 receptor in dorsal root ganglia (DRG). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in T2DM rats were lower than those of control rats. MWT and TWL in T2DM rats treated with sinomenine were higher compared with those in T2DM rats. The expression levels of the P2X3 protein and mRNA in T2DM rat DRG were higher compared with those of the control, while those in T2DM rats treated with sinomenine were significantly lower compared with those of the T2DM rats. Sinomenine significantly inhibited P2X3 agonist ATP-activated currents in HEK293 cells transfected with the P2X3 receptor. Sinomenine decreased the phosphorylation and activation of P38MAPK in T2DM DRG. Therefore, sinomenine treatment may suppress the up-regulated expression and activation of the P2X3 receptor and relieve the hyperalgesia potentiated by the activation of P38MAPK in T2DM rats.

Trans-Resveratrol Attenuates High Fatty Acid-Induced P2X7 Receptor Expression and IL-6 Release in PC12 Cells: Possible Role of P38 MAPK Pathway

ABSTRACTDiabetic neuropathy (DNP) is the most common chronic complication of diabetes. Elevated free fatty acids (FFAs) have been recently recognized as major causes of inflammation and are relevant to the functional changes of nerve system in diabetes. Trans-resveratrol (RESV), a polyphenolic natural compound, has long been acknowledged to have anti-inflammation properties and may exert a neuroprotective effect on neuronal damage in diabetes, while the mechanisms underlying are largely unknown. Our previous study on differential PC12 cells cultured with high FFAs has shown chronic FFAs overload increased PC12 interleukin (IL)-6 release mediated by P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine triphosphate (ATP); a high FFA-induced activation of P38 mitogen-activated protein kinase (MAPK) pathway was pointed to be a potential underlying mechanism. Data from this study indicated that RESV, in a dose-dependent manner, reduced high FFA-induced IL-6 release by impeding the activation of P2X7 receptor, as shown by the results that both high FFA-elevated P2X7 receptor messenger RNA (mRNA) and protein expression as well as high FFA-evoked [Ca2+]i in response to 3′-O-(4-benzoyl) benzoyl-ATP (a selective P2X7 receptor agonist) were significantly attenuated. Meanwhile, high FFA-induced activation of P38 MAPK, an essential prerequisite for high FFA-activated P2X7 receptor and subsequent IL-6 release, was also dose-dependently abrogated by RESV. Furthermore, RESV may hamper the activation of P38a MAPK (one paramount P38 isoform) via forming hydrogen bonding with Thr175 residue, surrounding the two residues (Thy180 and Tyr182) essential for canonical activation of P38a MAPK. Taken together, RESV could inhibit high FFA-induced inflammatory IL-6 release mediated by P2X7 receptor through deactivation of P38 MAPK signaling pathway. All these results outline the potential mechanisms involved in the neuroprotective roles of RESV and highlight the clinical application of RESV in treatment of inflammation in relation to DNP.

Long noncoding NONRATT021972 siRNA normalized abnormal sympathetic activity mediated by the upregulation of P2X7 receptor in superior cervical ganglia after myocardial ischemia

Previous studies showed that the upregulation of the P2X7 receptor in cervical sympathetic ganglia was involved in myocardial ischemic (MI) injury. The dysregulated expression of long noncoding RNAs (lncRNAs) participates in the onset and progression of many pathological conditions. The aim of this study was to investigate the effects of a small interfering RNA (siRNA) against the NONRATT021972 lncRNA on the abnormal changes of cardiac function mediated by the up-regulation of the P2X7 receptor in the superior cervical ganglia (SCG) after myocardial ischemia. When the MI rats were treated with NONRATT021972 siRNA, their increased systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), low-frequency (LF) power, and LF/HF ratio were reduced to normal levels. However, the decreased high-frequency (HF) power was increased. GAP43 and tyrosine hydroxylase (TH) are markers of nerve sprouting and sympathetic nerve fibers, respectively. We found that the TH/GAP43 value was significantly increased in the MI group. However, it was reduced after the MI rats were treated with NONRATT021972 siRNA. The serum norepinephrine (NE) and epinephrine (EPI) concentrations were decreased in the MI rats that were treated with NONRATT021972 siRNA. Meanwhile, the increased P2X7 mRNA and protein levels and the increased p-ERK1/2 expression in the SCG were also reduced. NONRATT021972 siRNA treatment inhibited the P2X7 agonist BzATP-activated currents in HEK293 cells transfected with pEGFP-P2X7. Our findings suggest that NONRATT021972 siRNA could decrease the upregulation of the P2X7 receptor and improve the abnormal changes in cardiac function after myocardial ischemia.

The protective effect of resveratrol in the transmission of neuropathic pain mediated by the P2X7 receptor in the dorsal root ganglia

&NA; The P2X7 receptor mediates afferent nerve activation and is related to chronic neuropathic pain. Resveratrol (RES) has also been reported to exhibit anti‐inflammatory effects. In this study, we investigated the neuroprotective effect of RES on the transmission of neuropathic pain mediated by the P2X7 receptor. The P2X7 mRNA and protein expression levels in L4‐L5 dorsal root ganglia (DRG)s of the chronic constriction injury (CCI) group were significantly higher than those observed in the Ctrl + NS, Sham + RES and Sham groups. RES increased the threshold of thermal and mechanical hypersensitivity in rats with chronic neuropathic pain. The P2X7 mRNA and protein expression levels in the CCI + RES group were decreased compared with those in the CCI group. Our results showed that RES inhibited the upregulated co‐expression of P2X7 and glial fibrillary acidic protein (GFAP, a marker of satellite glial cells) in satellite glial cells of DRG in the CCI group. The results demonstrated that the expression of GFAP was increased in the CCI group and that RES inhibited the upregulated expression of GFAP in the rats in the CCI group. In addition, the phosphorylation levels of p38 and extracellular regulated protein kinases (ERK)1/2 in the CCI group were markedly higher than those observed in the Ctrl + NS, Sham + RES and Sham groups, whereas the phosphorylation levels of p38 and ERK1/2 in CCI + RES group were markedly lower than those observed in the CCI group. RES inhibited BzATP‐activated currents in DRG non‐neurons in the CCI rats. Our data provide evidence that RES may suppress the transmission of neuropathic pain mediated by the P2X7 receptor in the satellite glial cells of dorsal root ganglia. HighlightsActivated P2X7 receptor in DRG SGC aggravated pain in CCI rats.Upregulation of P2X7 and GFAP, cytokines in DRG was increased after CCI.RES inhibited BzATP‐activated currents in DRG non‐neurons in CCI rats.RES inhibited upregulated P2X7 and GFAP, cytokines in DRG.RES relieved hyperalgesia mediated by P2X7 in DRG SGCs of CCI rats.

LncRNA uc.48+ siRNA improved diabetic sympathetic neuropathy in type 2 diabetic rats mediated by P2X7 receptor in SCG

Diabetic autonomic neuropathy includes the sympathetic ganglionic dysfunction. P2X7 receptor in superior cervical ganglia (SCG) participated in the pathological changes of cardiac dysfunction. Abnormal expression of long noncoding RNAs (lncRNAs) was reported to be involved in nervous system diseases. Our preliminary results obtained from rat lncRNA array profiling revealed that the expression of the uc.48+ was significantly increased in the rat SCG in response to diabetic sympathetic pathology. In this study, we found that lncRNAuc.48+ and P2X7 receptor in the SCG were increased in type 2 diabetic rats and were associated with the cardiac dysfunction. The uc.48+ small interference RNA (siRNA) improved the cardiac autonomic dysfunction and decreased the up-regulation P2X7 and the ratio of phosphorylated extracellular regulated protein kinases1/2 (p-ERK1/2) to ERK1/2 in SCG of type 2 diabetic rats. In conclusion, lncRNA uc.48+ siRNA improved diabetic sympathetic neuropathy in type 2 diabetic rats through regulating the expression of P2X7 and ERK signaling in SCG.