Activation of HCN channels caused by elevated cAMP levels in periaqueductal gray promotes bone cancer pain.

The periaqueductal gray (PAG) is an important relay center for the descending pathways that regulate nociceptive information transduction. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the nerve injury-induced pain hypersensitivity. Previous studies have identified that HCN1 and HCN2 channel protein located in the ventral-lateral periaqueductal gray (vlPAG), a region important for pain regulation. However, it is not clear whether the HCN channel in vlPAG is involved in bone cancer pain (BCP). In this study, we assessed the role of HCN channels in BCP by measuring changes of HCN channel expression and activity in vlPAG neurons in bone cancer rats. In the present study, the BCP model was established by injecting SHZ-88 breast cancer cells into the right tibia bone marrow in rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to evaluate pain behavior in rats. HCN1 and HCN2 channels expression in vlPAG were detected by using Western Blot and immunohistochemistry. In addition, the cAMP level in vlPAG neurons was detected by ELISA, and HCN channel current (Ih) of vlPAG neurons was recorded by whole cell patch-clamp to evaluate HCN channel activity. As a result, decreased MWT and TWL were observed in rats on 7d after SHZ-88 cell inoculation, and the allodynia was sustained until 21d after inoculation. At the same time, HCN1 and HCN2 channels expression and neuronal Ih in vlPAG were significantly increased in BCP rats. In addition, the level of cAMP in vlPAG also increased after SHZ-88 cell inoculation. Furthermore, intravlPAG injection of ZD7288 (HCN channels antagonist) could significantly reduce hyperalgesia and the elevation of cAMP in vlPAG in BCP rats. Our observations suggest that the elevation of cAMP may promote the activation of HCN channels in vlPAG in bone cancer rats, thereby promoting the development of bone cancer pain.

MiR-29c Inhibits TNF-α-Induced ROS Production and Apoptosis in Mouse Hippocampal HT22 Cell Line.

Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. However, the underlying effect of miR-29c in regulating hippocampal neuronal function is 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 administration. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Hoechst 33258 staining and TUNEL assay were used to evaluate cell apoptosis. The expression of key mRNA/proteins (TNFR1, Bcl-2, Bax, TRADD, FADD, caspase-3, -8 and -9) in the apoptosis pathway was detected by PCR or WB. In addition, the protein expression of microtubule-associated protein-2 (MAP-2), nerve growth-associated protein 43 (GAP-43) and synapsin-1 (SYN-1) was detected by WB. As a result, we found that miR-29c overexpression could improve cell viability, attenuate LDH release, reduce ROS production and inhibit MMP depolarization in TNF-α-treated HT22 cells. Furthermore, miR-29c overexpression was found to decrease apoptotic rate, along with decreased expression of Bax, cleaved caspase-3, cleaved caspase-9, and increased expression of Bcl-2 in TNF-α-treated HT22 cells. However, miR-29c sponge exhibited an opposite effects. In addition, in TNF-α-treated HT22 cells, miR-29c overexpression could decrease the expressions of TNFR1, TRADD, FADD and cleaved caspase-8. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced the expressions of TNFR1, TRADD, FADD and cleaved caspase-8 was significantly exacerbated. At last, TNF-α-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was reversed by miR-29c but exacerbated by miR-29c sponge. Overall, our study demonstrated that miR-29c protects against TNF-α-induced HT22 cells injury through alleviating ROS production and reduce neuronal apoptosis. Therefore, miR-29c might be a potential therapeutic agent for TNF-α accumulation and toxicity-related brain diseases.

Blockage of HCN Channels Inhibits the Function of P2X Receptors in Rat Dorsal Root Ganglion Neurons.

Hyperpolarization-activated cyclic nucleotide-gated channels and purinergic P2X receptors play critical roles in the nerve injury-induced pain hypersensitivity. Both HCN channels and P2XR are expressed in dorsal root ganglia sensory neurons. However, it is not clear whether the expression and function of P2X2 and P2X3 receptors can be modulated by HCN channel activity. For this reason, in rats with chronic constriction injury of sciatic nerve, we evaluated the effect of intrathecal administration of HCN channel blocker ZD7288 on nociceptive behavior and the expression of P2X2 and P2X3 in rat DRG. The mechanical withdrawal threshold was measured to evaluate pain behavior in rats. The protein expression of P2X2 and P2X3 receptor in rat DRG was observed by using Western Blot. The level of cAMP in rat DRG was measured by ELISA. As a result, decreased MWT was observed in CCI rats on 1 d after surgery, and the allodynia was sustained throughout the experimental period. In addition, CCI rats presented increased expression of P2X2 and P2X3 receptor in the ipsilateral DRG at 7 d and 14 d after CCI operation. Intrathecal injection of ZD7288 significantly reversed CCI-induced mechanical hyperalgesia, and attenuated the increased expression of P2X2 and P2X3 receptor in rat DRG, which open up the possibility that the expression of P2X2 and P2X3 receptor in DRG is down-regulated by HCN channel blocker ZD7288 in CCI rats. Furthermore, the level of cAMP in rat DRG significantly increased after nerve injury. Intrathecal administration of ZD7288 attenuated the increase of cAMP in DRG caused by nerve injury. Subsequently, effects of HCN channel activity on ATP-induced current (IATP) in rat DRG neurons were explored by using whole-cell patch-clamp techniques. ATP (100 µM) elicited three types of currents (fast, slow and mixed IATP) in cultured DRG neurons. Pretreatment with ZD7288 concentration-dependently inhibited three types of ATP-activated currents. On the other hand, pretreatment with 8-Br-cAMP (a cell-permeable cAMP analog, also known as an activator of PKA) significantly increased the amplitude of fast, slow and mixed IATP in DRG neurons. The enhanced effect of 8-Br-cAMP on ATP-activated currents could be reversed by ZD7288. In a summary, our observations suggest that the opening of HCN channels could enhance the expression and function of P2X2 and P2X3 receptor via the cAMP-PKA signaling pathway. This may be important for pathophysiological events occurring within the DRG, for where it is implicated in nerve injury-induced pain hypersensitivity.

CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 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 Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 µM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ 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 [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ 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 Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 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 CB1R rather than CB2R 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 CB1R on P2X-channel-induced Ca2+ mobilization.

Preparation and evaluation of sustained-release solid dispersions co-loading gastrodin with borneol as an oral brain-targeting enhancer.

Borneol is a traditional Chinese medicine that can promote drug absorption from the gastrointestinal tract and distribution to the brain. However, stomach irritation may occur when high doses of borneol are used. In the present work, gastrodin, the main bioactive ingredient of the traditional Chinese drug "Tianma" (Rhizoma Gastrodiae) was used as a model drug to explore reasonable application of borneol. Sustained-release solid dispersions (SRSDs) for co-loading gastrodin and borneol were prepared using ethylcellulose as a sustained release matrix and hydroxy-propyl methylcellulose as a retarder. The dispersion state of drug within the SRSDs was analyzed by using scanning electron microscopy, differential scanning calorimetry, and powder X-ray diffractometry. The results indicated that both gastrodin and borneol were molecularly dispersed in an amorphous form. Assay of in vitro drug release demonstrated that the dissolution profiles of gastrodin and borneol from the SRSDs both fitted the Higuchi model. Subsequently, gastric mucosa irritation and the brain targeting of the SRSDs were evaluated. Compared with the free mixture of gastrodin and borneol, brain targeting of SRSDs was slightly weaker (brain targeting index: 1.83 vs. 2.09), but stomach irritation obviously reduced. Sustained-release technology can be used to reduce stomach irritation caused by borneol while preserving sufficient transport capacity for oral brain-targeting drug delivery.

Role of glucose transporters in the intestinal absorption of gastrodin, a highly water-soluble drug with good oral bioavailability.

Gastrodin, a sedative drug, is a highly water-soluble phenolic glucoside with poor liposolubility but exhibits good oral bioavailability. The current study aims to investigate whether glucose transporters (GLTs) are involved in the intestinal absorption of gastrodin. The intestinal absorption kinetics of gastrodin was determined using the rat everted gut sac model, the Caco-2 cell culture model and the perfused rat intestinal model. In vivo pharmacokinetic studies using diabetic rats with high GLT expression were performed. Saturable intestinal absorption of gastrodin was observed in rat everted gut sacs. The apparent permeability (Papp) of gastrodin from the apical (A) to basolateral (B) side in Caco-2 cells was two-fold higher than that from B to A. Glucose or phlorizin, a sodium-dependent GLT (SGLT) inhibitor, reduced the absorption rates of gastrodin from perfused rat intestines. In vivo pharmacokinetic studies showed that the time of maximum plasma gastrodin concentration (Tmax) was prolonged from 28 to 72 min when orally co-administered with four times higher dose of glucose. However, the Tmax of gastrodin in diabetic rats was significantly lowered to 20 min because of the high intestinal SGLT1 level. In conclusion, our findings indicate that SGLT1 can facilitate the intestinal absorption of gastrodin.