Activation of 5-HT6 Receptors in the Ventrolateral Orbital Cortex Produces Anti-Anxiodepressive Effects in a Rat Model of Neuropathic Pain.

The comorbidity of anxiety and depression frequently occurs in patients with neuropathic pain. The ventrolateral orbital cortex (VLO) plays a critical role in mediating neuropathic pain and anxiodepression in rodents. Previous studies suggested that 5-HT6 receptors in the VLO are involved in neuropathic pain. Strong evidence supports a close link between 5-HT6 receptors and affective disorders such as depression and anxiety disorders. However, it remains unclear whether the 5-HT6 receptors in the VLO are involved in neuropathic pain-induced anxiodepression. Using a rat neuropathic pain model of spared nerve injury (SNI), we demonstrated that rats exhibited significant anxiodepression-like behaviors and the expression of VLO 5-HT6 receptors obviously decreased four weeks after SNI surgery. Microinjection of the 5-HT6 receptor agonist EMD-386088 into the VLO or overexpression of VLO 5-HT6 receptors alleviated anxiodepression-like behaviors. These effects were blocked by pre-microinjection of a selective 5-HT6 receptor antagonist (SB-258585) or inhibitors of AC (SQ-22536), PKA (H89), and MEK1/2 (U0126) respectively. Meanwhile, the expression of p-ERK, p-CREB, and BDNF in the VLO decreased four weeks after SNI surgery. Furthermore, administration of EMD-386088 upregulated the expression of BDNF, p-ERK, and p-CREB in the VLO of SNI rats, which were reversed by pre-injection of SB-258585. These findings suggest that activating 5-HT6 receptors in the VLO has anti-anxiodepressive effects in rats with neuropathic pain via activating AC-cAMP-PKA-MERK-CREB-BDNF signaling pathway. Accordingly, 5-HT6 receptor in the VLO could be a potential target for the treatment of the comorbidity of neuropathic pain and anxiodepression.

Activation of 5-HT5A receptor in the ventrolateral orbital cortex produces antinociceptive effects in rat models of neuropathic and inflammatory pain.

The ventrolateral orbital cortex (VLO) is identified as an integral component of the endogenous analgesic system comprising a spinal cord - thalamic nucleus submedius - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study investigates the effects of 5-HT5A receptor activation in the VLO on allodynia induced by spared nerve injury and formalin-evoked flinching behavior and spinal c-Fos expression in male SD rats, and further examines whether GABAergic modulation is involved in the effects evoked by VLO 5-HT5A receptor activation. We found an upregulation of 5-HT5A receptor expression in the VLO during neuropathic and inflammatory pain states. Microinjection of the non-selective 5-HT5A receptor agonist 5-CT into the VLO dose dependently alleviated allodynia, and flinching behavior and spinal c-Fos expression, which were blocked by the selective 5-HT5A receptor antagonist SB-699551. Moreover, application of the GABAA receptor antagonist bicuculline in the VLO augmented the analgesic effects induced by 5-CT in neuropathic and inflammatory pain states, whereas the GABAA receptor agonist muscimol attenuated these analgesic effects. Additionally, the 5-HT5A receptors were found to be colocalized with GABAergic neurons in the VLO. These results provide new evidence for the involvement of central 5-HT5A receptors in the VLO in modulation of neuropathic and inflammatory pain and support the hypothesis that activation of 5-HT5A receptors may inhibit the inhibitory effect of GABAergic interneurons on output neurons projecting to the PAG (GABAergic disinhibitory mechanisms), consequently activating the brainstem descending inhibitory system that depresses nociceptive transmission at the spinal cord level.

CD147-specific chimeric antigen receptor T cells effectively inhibit T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice were used to establish a T-ALL xenograft model and CD147-CAR T cells conferred robust protection against T-ALL progression and significantly improved survival in mice. Overall, we found that CD147 is a potential antigen target of CAR T cell therapy for T-ALL.

Spinal 5-HT2A receptor is involved in electroacupuncture inhibition of chronic pain.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, and it is a typical disease which can develop chronic pain. Our previous study has proved that endocannabinoid (2-AG)-CB1R-GABA-5-HT pathway is involved in electroacupuncture (EA) mediated inhibition of chronic pain. However, it is still unclear which among the 5-HT receptor subtype is involved in EA evoked 5-HT mediated inhibition of chronic pain in the dorsal spinal cord. 5-HT2A is a G protein-coupled receptor and it is involved in 5-HT descending pain modulation system. We found that EA treatment at frequency of 2 Hz +1 mA significantly increased the expression of 5-HT2A receptor in the dorsal spinal cord and intrathecal injection of 5-HT2A receptor antagonist or agonist reversed or mimicked the analgesic effect of EA in each case respectively. Intrathecal injection of a selective GABAA receptor antagonist Bicuculline also reversed the EA effect on pain hypersensitivity. Additionally, EA treatment reversed the reduced expression of GABAA receptor and KCC2 in the dorsal spinal cord of KOA mice. Furthermore, we demonstrated that intrathecal 5-HT2A receptor antagonist/agonist reversed or mimicked the effect of EA up-regulate of KCC2 expression, respectively. Similarly, intrathecal injection of PLC and PKC inhibitors prevented both anti-allodynic effect and up-regulation of KCC2 expression by EA treatment. Our data suggest that EA treatment up-regulated KCC2 expression through activating 5-HT2A-Gq-PLC-PKC pathway and enhanced the inhibitory function of GABAA receptor, thereby inhibiting chronic pain in a mouse model of KOA.

Active demethylation upregulates CD147 expression promoting non-small cell lung cancer invasion and metastasis.

Non-small cell lung cancer (NSCLC) is a fatal disease, and its metastatic process is poorly understood. Although aberrant methylation is involved in tumor progression, the mechanisms underlying dynamic DNA methylation remain to be elucidated. It is significant to study the molecular mechanism of NSCLC metastasis and identify new biomarkers for NSCLC early diagnosis. Here, we performed MeDIP-seq and hMeDIP-seq analyses to detect the genes regulated by dynamic DNA methylation. Comparison of the 5mC and 5hmC sites revealed that the CD147 gene underwent active demethylation in NSCLC tissues compared with normal tissues, and this demethylation upregulated CD147 expression. Significantly high levels of CD147 expression and low levels of promoter methylation were observed in NSCLC tissues. Then, we identified the CD147 promoter as a target of KLF6, MeCP2, and DNMT3A. Treatment of cells with TGF-ß triggered active demethylation involving loss of KLF6/MeCP2/DNMT3A and recruitment of Sp1, Tet1, TDG, and SMAD2/3 transcription complexes. A dCas9-SunTag-DNMAT3A-sgCD147-targeted methylation system was constructed to reverse CD147 expression. The targeted methylation system downregulated CD147 expression and inhibited NSCLC proliferation and metastasis in vitro and in vivo. Accordingly, we used cfDNA to detect the levels of CD147 methylation in NSCLC tissues and found that the CD147 methylation levels exhibited an inverse relationship with tumor size, lymphatic metastasis, and TNM stage. In conclusion, this study clarified the mechanism of active demethylation of CD147 and suggested that the targeted methylation of CD147 could inhibit NSCLC invasion and metastasis, providing a highly promising therapeutic target for NSCLC.

Ionizing Radiation-Induced RPL23a Reduction Regulates Apoptosis via RPL11-MDM2-p53 Pathway in Mouse Spermatogonia.

OBJECTIVE: The expression patterns of ribosomal large subunit protein 23a (RPL23a) in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23a expression and spermatogonia apoptosis upon exposure to X-ray. METHODS: Male mice and GC-1 cells were irradiated with X-ray, terminal dUTP nick end-labelling (TUNEL) was performed to detect apoptotic spermatogonia in vivo. Apoptotic rate and cell cycle phase of GC-1 cells were analyzed with flow cytometry. Protein interactions were detected by Immunoprecipitation and protein localization as studied by immunofluorescence. Immunoblotting and real-time PCR were applied to analyze to protein and gene expression. RESULTS: Ionizing radiation (IR) increased spermatogonia apoptosis, the expression of RPL11, MDM2 and p53, and decreased RPL23a expression in mice spermatogonia in vivo and in vitro. RPL23a knockdown weakened the interaction between RPL23a and RPL11, leading to p53 accumulation. Moreover, knockdown and IR decreased RPL23a that induces spermatogonia apoptosis via RPL23a-RPL11-MDM2-p53 pathway in GC-1 cells. CONCLUSION: These results suggested that IR reduced RPL23a expression, leading to weakened the RPL23a-RPL11 interactions, which may have activated p53, resulting in spermatogonia apoptosis. These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility. The graphical abstract was available in the web of www.besjournal.com.

5-HT7 Receptor Is Involved in Electroacupuncture Inhibition of Chronic Pain in the Spinal Cord.

Knee osteoarthritis (KOA) is a common and disabling condition characterized by attacks of pain around the joints, and it is a typical disease that develops chronic pain. Previous studies have proved that 5-HT1, 5-HT2, and 5-HT3 receptors in the spinal cord are involved in electroacupuncture (EA) analgesia. The 5-HT7 receptor plays antinociceptive role in the spinal cord. However, it is unclear whether the 5-HT7 receptor is involved in EA analgesia. The 5-HT7 receptor is a stimulatory G-protein (Gs)-coupled receptor that activates adenylyl cyclase (AC) to stimulate cyclic adenosine monophosphate (cAMP) formation, which in turn activates protein kinase A (PKA). In the present study, we found that EA significantly increased the tactile threshold and the expression of the 5-HT7 receptor in the dorsal spinal cord. Intrathecal injection of 5-HT7 receptor agonist AS-19 mimicked the analgesic effect of EA, while a selective 5-HT7 receptor antagonist reversed this effect. Moreover, intrathecal injection of AC and PKA antagonists prior to EA intervention prevented its anti-allodynic effect. In addition, GABAA receptor antagonist bicuculline administered (intrathecal, i.t.) prior to EA intervention blocked the EA effect on pain hypersensitivity. Our data suggest that the spinal 5-HT7 receptor activates GABAergic neurons through the Gs-cAMP-PKA pathway and participates in EA-mediated inhibition of chronic pain in a mouse model of KOA.