Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some nervous system disease. However, many aspects underlying mechanism of miR-29c in regulating cellular functions in hippocampal neurons are not clear. In this study, HT22 cells were infected with lentivirus containing miR-29c or miR-29c sponge. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay kit were applied to evaluate cell viability and toxicity before and after TNF-α application. The intracellular reactive oxygen species (ROS) concentration were evaluated using dihydroethidium (DHE) staining and the mitochondrial membrane potential (MMP) was determined using Tetramethylrhodamine methyl ester (TMRM) staining. Cellular apoptosis was observed via TUNEL staining assay and Hoechst 33258 staining and measured by the expression of cleaved caspase-3, -8 and − 9. The expression of miR-29c and TNFR1, Bcl-2, Bax, TRADD and FADD was analyzed by PCR or WB. As a result, we found that miR-29c overexpression signi cantly attenuated decreases in cell viability, release of LDH and apoptosis in TNF-α-treated HT22 cells. Furthermore, the high expression of miR-29c can signi cantly prevented TNF-α-induced oxidative injuries via the decrease of intracellular ROS levels and the increase of MMP. TNF-α-induced increase in Bax and cleaved-caspase-9 and the decrease of Bcl-2 was attenuated after transfection of HT22 cells with miR-29c. Moreover, over-expression of miR-29c signi cantly inhibits the increased expression of TNFR1, TRADD, FADD, cleaved caspase-8 and − 3. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced cytotoxicity and oxidative stress, as well as elevated expression of TNFR1, TRADD and FADD were exacerbated. Our ndings indicated that miR-29c overexpression attenuates TNF-α-induced HT22 cells injury through inhibiting mitochondrial oxidative stress-induced apoptosis. In addition, TNF-αinduced TNFR1 activation, FADD association and caspase 8 recruitment may be impaired by miR-29c. Therefore, miR-29c might be a potential therapeutic target for the treatment of TNF-α-induced neurotoxicity.
Spinal cannabinoid receptor 1 (CBR) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CBR and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca]i increase in cultured DH neurons. ATP-evoked [Ca]i increase in DH neurons was blocked by chelating extracellular Ca and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca response was mimicked by ACEA (CBR agonist), but was not influenced by AM1241 (CBR agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca mobilization was blocked by AM251 (CB receptor antagonist), but was not influenced by AM630 (CB receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CBR rather than CBR can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CBR on P2X-channel-induced Ca mobilization.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.