MiR-146a alleviates inflammatory bowel disease in mice through systematic regulation of multiple genetic networks.

Introduction: Inflammatory bowel disease (IBD) is a chronic disease involving multiple genes, and the current available targeted drugs for IBD only deliver moderate efficacy. Whether there is a single gene that systematically regulates IBD is not yet known. MiR-146a plays a pivotal role in repression of innate immunity, but its function in the intestinal inflammation is sort of controversy, and the genetic regulatory networks regulated by miR-146a in IBD has not been revealed. Methods: RT-qPCR was employed to detect the expression of miR-146a in IBD patients and in a mouse IBD model induced by dextran sulfate sodium (DSS), and then we generated a miR-146a knock-out mouse line with C57/Bl6N background. The disease activity index was scored in DSS-treated miR-146a deficiency mice and their wild type (WT) littermates. Bulk RNA-sequencing, RT-qPCR and immunostaining were done to illustrate the downstream genetic regulatory networks of miR-146a in flamed colon. Finally, the modified miR-146a mimics were used to treat DSS-induced IBD in miR-146a knock-out and WT IBD mice. Results: We showed that the expression of miR-146a in the colon was elevated in dextran sulfate sodium (DSS)-induced IBD mice and patients with IBD. DSS induced dramatic body weight loss and more significant rectal bleeding, shorter colon length, and colitis in miR-146a knock-out mice than WT mice. The miR-146a mimics alleviated DSS-induced symptoms in both miR-146a-/- and WT mice. Further RNA sequencing illustrated that the deficiency of miR-146a de-repressed majority of DSS-induced IBD-related genes that cover multiple genetic regulatory networks in IBD, and supplementation with miR-146a mimics inhibited the expression of many IBD-related genes. Quantitative RT-PCR or immunostaining confirmed that Ccl3, Saa3, Csf3, Lcn2, Serpine1, Serpine2, MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, P65, p-P65, and IRAK1 were regulated by miR-146a in DSS induced IBD. Among them, MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, CSF3, and IL1A were involved in the active stage of IBD in humans. Discussion: Our date demonstrated that miR-146a acts as a top regulator in C57/BL6N mice to systematically repress multiple genetic regulatory networks involved in immune response of intestine to environment factors, and combinatory treatment with miR-146a-5p and miR-146a-3p mimics attenuates DSS-induced IBD in mice through down-regulating multiple genetic regulatory networks which were increased in colon tissue from IBD patients. Our findings suggests that miR-146a is a top inhibitor of IBD, and that miR-146a-5p and miR-146a-3p mimics might be potential drug for IBD.

Ectopic expression of Nav1.7 in spinal dorsal horn neurons induced by NGF contributes to neuropathic pain in a mouse spinal cord injury model.

Neuropathic pain (NP) induced by spinal cord injury (SCI) often causes long-term disturbance for patients, but the mechanisms behind remains unclear. Here, our study showed SCI-induced ectopic expression of Nav1.7 in abundant neurons located in deep and superficial laminae layers of the spinal dorsal horn (SDH) and upregulation of Nav1.7 expression in dorsal root ganglion (DRG) neurons in mice. Pharmacologic studies demonstrated that the efficacy of the blood-brain-barrier (BBB) permeable Nav1.7 inhibitor GNE-0439 for attenuation of NP in SCI mice was significantly better than that of the BBB non-permeable Nav1.7 inhibitor PF-05089771. Moreover, more than 20% of Nav1.7-expressing SDH neurons in SCI mice were activated to express FOS when there were no external stimuli, suggesting that the ectopic expression of Nav1.7 made SDH neurons hypersensitive and Nav1.7-expressing SDH neurons participated in central sensitization and in spontaneous pain and/or walking-evoked mechanical pain. Further investigation showed that NGF, a strong activator of Nav1.7 expression, and its downstream JUN were upregulated after SCI in SDH neurons with similar distribution patterns and in DRG neurons too. In conclusion, our findings showed that the upregulation of Nav1.7 was induced by SCI in both SDH and DRG neurons through increased expression of NGF/JUN, and the inhibition of Nav1.7 in both peripheral and spinal neurons alleviated mechanical pain in SCI mice. These data suggest that BBB permeable Nav1.7 blockers might relieve NP in patients with SCI and that blocking the upregulation of Nav1.7 in the early stage of SCI via selective inhibition of the downstream signaling pathways of NGF or Nav1.7-targeted RNA drugs could be a strategy for therapy of SCI-induced NP.

MicroRNA-96 is required to prevent allodynia by repressing voltage-gated sodium channels in spinal cord.

Voltage-gated sodium channels (Navs) 1.7, 1.8, and 1.9 are predominately expressed in peripheral sensory neurons and are critical for action potential propagation in nociceptors. Unexpectedly, we found that expression of SCN9A, SCN10A, SCN11A, and SCN2A, the alpha subunit of Nav1.7, Nav1.8, Nav1.9 and Nav1.2, respectively, are up-regulated in spinal dorsal horn (SDH) neurons of miR-96 knockout mice. These mice also have de-repression of CACNA2D1/2 in DRG and display thermal and mechanical allodynia that could be attenuated by intrathecal or intraperitoneal injection of Nav1.7 or Nav1.8 blockers or Gabapentin. Moreover, Gad2::CreERT2 conditional miR-96 knockout mice phenocopied global knockout mice, implicating inhibitory neurons; nerve injury induced significant loss of miR-96 in SDH GABAergic and Glutamatergic neurons in mice which negatively correlated to up-regulation of Nav1.7, Nav1.8, Nav1.9 and Scn2a, this dis-regulation of miR-96 and Navs in SDH neurons contributed to neuropathic pain which can be alleviated by intrathecal injection of Nav1.7 or Nav1.8 blockers. In conclusion, miR-96 is required to avoid allodynia through limiting the expression of VGCCs and Navs in DRG and Navs in SDH in naïve and nerve injury-induced neuropathic pain mice. Our findings suggest that central nervous system penetrating Nav1.7 and Nav1.8 blockers may be efficacious for pain relief.

Hemokinin-1(4-11)-induced analgesia selectively up-regulates δ-opioid receptor expression in mice.

Our previous studies have shown that an active fragment of human tachykinins (hHK-1(4-11)) produced an opioid-independent analgesia after intracerebroventricular (i.c.v.) injection in mice, which has been markedly enhanced by a δ OR antagonist, naltrindole hydrochloride (NTI). In this study, we have further characterized the in vivo analgesia after i.c.v. injection of hHK-1(4-11) in mouse model. Our qRT-PCR results showed that the mRNA levels of several ligands and receptors (e.g. PPT-A, PPT-C, KOR, PDYN and PENK) have not changed significantly. Furthermore, neither transcription nor expression of NK1 receptor, MOR and POMC have changed noticeably. In contrast, both mRNA and protein levels of DOR have been up-regulated significantly, indicating that the enhanced expression of δ opioid receptor negatively modulates the analgesia induced by i.c.v. injection of hHK-1(4-11). Additionally, the combinatorial data from our previous and present experiments strongly suggest that the discriminable distribution sites in the central nervous system between hHK-1(4-11) and r/mHK-1 may be attributed to their discriminable analgesic effects. Altogether, our findings will not only contribute to the understanding of the complicated mechanisms regarding the nociceptive modulation of hemokinin-1 as well as its active fragments at supraspinal level, but may also lead to novel pharmacological interventions.

Study on the distribution sites and the molecular mechanism of analgesia after intracerebroventricular injection of rat/mouse hemokinin-1 in mice.

Hemokinin-1 is a peptide encoded by Pptc, which belongs to the family of mammalian tachykinins. Our previous results showed that rat/mouse hemokinin-1 (r/m HK-1) produced striking analgesia after intracerebroventricular (i.c.v.) injection in mice, and the analgesia could be blocked by the NK1 receptor antagonist and the opioid receptor antagonist, respectively. However, the precise distribution sites and the molecular mechanism involved in the analgesic effect after i.c.v. administration of r/m HK-1 are needed to be further investigated deeply. Using the fluorescence labeling method, our present results directly showed that r/m HK-1 peptides were mainly distributed at the ventricular walls and several juxta-ventricular structures for the first time. Our results showed that the mRNA expressions of NK1 receptor, PPT-A, PPT-C, KOR, PDYN, DOR and PENK were not changed markedly, as well as the protein expression of NK1 receptor was hardly changed. However, both the transcripts and proteins of MOR and POMC were up-regulated significantly, indicating that the analgesic effect induced by i.c.v. administration of r/m HK-1 is related to the activation of NK1 receptor first, then it is related to the release of endogenous proopiomelanocortin, as well as the increased expression level of µ opioid receptor. These results should facilitate further the analysis of the analgesia of r/m HK-1 in the central nerval system in acute pain and may open novel pharmacological interventions.

Different regulatory pathways are involved in the proliferative inhibition of two types of leukemia cell lines induced by paclitaxel.

Paclitaxel, one of the broadest-spectrum anticancer agents, is currently being used in the treatment of patients with solid tumors. In the present study, we compared the effect of paclitaxel on two types of leukemia cells. Our results showed that paclitaxel could inhibit the proliferation of MEL and K562 cells in a dose- and time-dependent manner. The mechanism of proliferative inhibition in K562 cells treated by paclitaxel was related to the cell cycle arrest in the G2/M phase, as well as the induction of apoptosis. By contrast, MEL cells treated by paclitaxel showed significant characteristics of necrosis, which indicated that the mode of cell death induced by paclitaxel in these two types of leukemia cells differed. Advances in research of the cell cycle, apoptosis and necrosis will extend our understanding of the mechanisms of paclitaxel-induced cell death, particularly in leukemia cells. Further elucidation of the mechanisms of necrosis in MEL cells may expedite the development of improved paclitaxel-based regimens for cancer therapy.