Formononetin reverses Treg/Th17 imbalance in immune-mediated bone marrow failure mice by regulating the PI3K/Akt signaling pathway.

BACKGROUND: Severe aplastic anemia (SAA) is a syndrome of bone marrow failure which is life-threatening. Recent studies have demonstrated that CD4 + T cell subsets, including T regulatory (Treg) and T helper 17 (Th17) cells, play a pivotal role in the pathogenesis of SAA. Formononetin (FMN) is a natural compound extracted from the traditional Chinese medicine Huangqi, which has the ability to regulate the imbalance of Treg/Th17 cells in some inflammatory diseases. Nevertheless, the therapeutic effect of FMN in SAA has yet to be definitively established. Therefore, the objective of this research was to investigate the effect of FMN on SAA and elucidate its underlying mechanism. METHODS: In vivo experiments, the mice were divided into the following five groups: control, model, low-dose FMN, high-dose FMN, and positive control cyclosporine A group. The immune-mediated bone marrow failure (BMF) mouse model was established by the total body X-ray radiation and lymphocyte infusion. After 10 days of continuous administration of FMN, the numbers of Treg/Th17 cells in the bone marrow and spleen were assessed by flow cytometry. The protein expressions of PI3K/Akt pathway in the bone marrow and spleen was assessed by immunohistochemistry and western blotting. In vitro, the impact of FMN on the differentiation of naive CD4 + T cells into Treg cells was investigated by flow cytometry and ELISA. RESULTS: In comparison with the control group, the model group showed a reduction in bone marrow nucleated cells, a significant decrease in peripheral blood cells, and an altered CD8 + /CD4 + T cell ratio. These findings indicate the successful establishment of a mouse model of immune-mediated BMF. After FMN treatment, there were the increased levels of red blood cells and hemoglobin. In addition, FMN mitigated the bone marrow destruction and restored the CD8 + /CD4 + T cell ratio. Furthermore, in comparison with the control group, the model group showed the decreased levels of Treg cells and the increased levels of Th17 cells. After FMN treatment, there was a significantly increased number of Treg cells and a decreased number of Th17 cells. Additionally, FMN remarkably down-regulated the expression levels of PI3K and Akt proteins in immune-mediated BMF mice. CONCLUSIONS: FMN alleviates immune-mediated BMF by modulating the balance of Treg/Th17 cells through the PI3K/Akt signaling pathway.

Assessing NH300094, a novel dopamine and serotonin receptor modulator with cognitive enhancement property for treating schizophrenia.

Background: Schizophrenia is a serious psychiatric disorder that significantly affects the quality of life of patients. The objective of this study is to discover a novel antipsychotic candidate with highly antagonistic activity against both serotonin and dopamine receptors, demonstrating robust efficacy in animal models of positive, negative, and cognitive symptoms of schizophrenia. Methods: In the present study, we examined the activity of antipsychotic drug (NH300094) on 5-HT2A, 5-HT2C, 5-HT1A, 5-HT1B, 5-HT7, H1, M1, Alpha1A, D2L, D2S, Alpha2A, D3 receptor functional assay in vitro. In addition, multiple animal models, including dizocilpine (MK-801) induced hyper-locomotion; APO induced climbing; Conditioned Avoidance Response (CAR); DOI-Induced Head Twitch; Forced swimming test; Scopolamine induced cognitive impairment model, were used to verify the antipsychotic activity of NH300094 in preclinical. Results: In vitro functional assays have indicated that NH300094 is a potent antagonist of 5-HT receptors and dopamine receptors, with higher relative antagonistic activity against 5-HT2A receptor (5-HT2A IC50 = 0.47 nM) than dopamine receptors (D2L IC50 = 1.04 nM; D2S IC50 = 11.71 nM; D3 IC50 = 31.55 nM). Preclinical in vivo pharmacological study results showed that NH300094 was effective in multiple models, which is more extensive than the clinic drug Risperidone. Furthermore, the safety window for extrapyramidal side effects of NH300094 is significantly wider than that of Risperidone (For NH300094, mice catalepsy model ED50/ Mice MK-801 model ED50 = 104.6-fold; for Risperidone, mice catalepsy model ED50/ Mice MK-801 model ED50 = 12.9-fold), which suggests a potentially better clinical safety profile for NH300094. Conclusion: NH300094 is a novel potent serotonin and dopamine receptors modulator, which has good safety profile and therapeutic potential for the treatment of schizophrenia with cognition disorders.

G-quadruplex-deficient precursor hairpin probes for ultra-low background dual-mode detection of miRNAs.

Design of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics. Herein, we engineered a miRNA-21 responsive G-quadruplex-deficient precursor hairpin probe (PHP) to achieve dual-mode detection of fluorescent signal and colorimetric signal. Due to lack of complete G-quadruplex sequence, PHP becomes shorter in length, lower background signal and less interference. Based on the polymerase-driven amplification mechanism, in the presence of miRNAs, two simultaneous amplification reaction processes will occur in PHP: miRNA-based amplification process and endogenous amplification process along the 3' end. Due to the positional difference between the starting points of the two amplification processes, the orderly and efficient occurrence of the two amplification processes can be achieved. Based on an interesting concept, PHP can achieve high detection performance with only simple amplification cycles. In such a way, the detection limits for fluorescence and colorimetry were 2.93 fM and 8.81 fM, which would cover most of clinical qualitative and quantitative needs. Thus, the accurate quantitative and visual miRNA detection technology based on PHP is beneficial to carry out extensive disease screening and treatment monitoring in various complex occasions.

Chronic activation of Mas-related gene receptors (Mrg) reduces the potency of morphine-induced analgesia via PKC pathway in naive rats.

Mas oncogene-related gene receptors (Mrg) are uniquely distributed in small and medium cells of trigeminal and dorsal root ganglia (DRG). The physiological and pharmacological properties of Mrg are unknown. We have shown that intermittent activation of MrgC prevents and reverses morphine tolerance. Now we observed that intrathecal (i.t.) administration of the MrgC agonist bovine adrenal medulla 8-22 (BAM8-22, 3 nmol) for 3 and 6 days reduced the potency of morphine analgesia by 1.5 and 3.5 folds, respectively. Daily administration of BAM8-22 for 6 days also significantly decreased the tail flick latency. The administration of another MrgC agonist (Tyr6)-γ2-MSH-6-12 (MSH, 3 nmol) reduced morphine potency and the reduction was abolished following the co-administration of the protein kinase C (PKC) inhibitor chelerythrine chloride (CLT, 3 nmol). The chronic treatment with BAM8-22 or MSH increased the expression of PKC-gamma (PKCγ) in the cell membrane of spinal dorsal horn neurons and PKC-epsilon (PKCε) in the cell membrane and cytosol of DRG neurons. Moreover, the BAM8-22 treatment induced an increase in the expression of calcitonin gene-related peptide (CGRP) and neuronal nitric oxide synthase (nNOS) in small and medium cells in DRG. All of these responses were not seen when BAM8-22 or MSH was co-administered with the PKC inhibitor CLT (3 nmol) or GF-109203X (10 nmol). The present study suggested that the chronic activation of MrgC upregulated expressions of pronociceptive mediators via PKC signaling pathway leading to the suppression of antinociceptive property of morphine. These effects are opposite to those occurred when MrgC is activated acutely or moderately.

Peripheral Leptin Signaling Mediates Formalin-Induced Nociception.

Accumulating evidence suggests that obesity is associated with chronic pain. However, whether obesity is associated with acute inflammatory pain is unknown. Using a well-established obese mouse model induced by a high-fat diet, we found that: (1) the acute thermal pain sensory threshold did not change in obese mice; (2) the model obese mice had fewer nociceptive responses in formalin-induced inflammatory pain tests; restoring the obese mice to a chow diet for three weeks partly recovered their pain sensation; (3) leptin injection induced significant phosphorylation of STAT3 in control mice but not in obese mice, indicating the dysmodulation of topical leptin-leptin receptor signaling in these mice; and (4) leptin-leptin receptor signaling-deficient mice (ob/ob and db/db) or leptin-leptin receptor pathway blockade with a leptin receptor antagonist and the JAK2 inhibitor AG 490 in wild-type mice reduced their nociceptive responses in formalin tests. These results indicate that leptin plays a role in nociception induced by acute inflammation and that interference in the leptin-leptin receptor pathway could be a peripheral target against acute inflammatory pain.

Discovery of aminocyclohexene analogues as selective and orally bioavailable hNav1.7 inhibitors for analgesia.

hNav1.7 receives a lot of attention owing to its attractive mechanism of action in pain processing pathway. We have previously reported our design of a novel series of tetrahydropyridine analogues towards hNav1.7 selective inhibitors. Herein, we disclose further efforts to the optimization of hit compound (-)-6, which led to the identification of aminocyclohexene analogues (-)-9 and (-)-17 with good potency, high selectivity, and minimal CYP inhibition. Both compounds (-)-9 and (-)-17 demonstrated improved pharmacokinetic profiles in rats, and robust efficacy in rat formalin-induced nociception and spinal nerve ligation (SNL) models.

The discovery of tetrahydropyridine analogs as hNav1.7 selective inhibitors for analgesia.

hNav1.7 small molecular inhibitors have attracted lots of attention by its unique analgesic effect. Herein, we report the design and synthesis of a novel series of tetrahydropyridine analogs as hNav1.7 inhibitors for analgesia. Detail structural-activity relationship (SAR) studies were undertaken towards improving hNav1.7 activity, in vitro ADME, and in vivo PK profiles. These efforts resulted in the identification of compound (-)-15h, a highly potent and selective hNav1.7 inhibitor with good ADME and PK profiles.

[Analysis of CYP21A2 gene mutations in two families with 21-hydroxylase deficiency].

OBJECTIVE: To analyze CYP21A2 gene mutation in two families with 21-hydroxylase deficiency (21-OHD) and to explore the correlation between genotype and clinical phenotype. METHODS: Two patients with 21-OHD and their families were investigated. CYP21A2 gene mutation was analyzed by PCR and direct sequencing. RESULTS: The probands from family 1 and 2 have been respectively diagnosed with simple virilizing and non-classical 21-OHD. Both showed increased baseline serum 17hydroxyprogesterone, testosterone and adrenocorticotropic hormone (ACTH), but had no evidence of salt loss. Computer tomography revealed bilateral adrenal hyperplasia in both patients. After 1 year treatment, both had conceived successfully. DNA sequencing revealed that the proband of family 1 had compound heterozygous mutations for IVS2 13 A>G and Ile172Asn. Her father was heterozygous for Ile172Asn, whilst her mother and brother were heterozygous for IVS213A/C>G. In family 2, the proband was heterozygous for Arg341Trp and Gln318X. Her father, sister and nephew were heterozygous for Arg341Trp, whilst her mother was heterozygous for Gln318X. her brother and niece were non-affected. Carriers of single heterozygous mutations in both families had no clinical sign. CONCLUSION: In both families, the disease has been caused by compound heterozygous mutations, for which there has been a good genotype-phenotype agreement. Screening of CYP21A2 gene can facilitate both diagnosis and genetic counseling.

INCB38579, a novel and potent histamine H4 receptor small molecule antagonist with anti-inflammatory pain and anti-pruritic functions.

The histamine H4 receptor mediates several histamine-induced cellular functions of leukocytes, including cell migration and cytokine production. Recent studies suggest that histamine signaling through the histamine H4 receptor can also have anti-pruritic and anti-nociceptive functions. 1-(7-(2-amino-6-(4-methylpiperazin-1-yl) pyrimidin-4-yl)-3, 4-dihdroisoquinolin-2(1H)-yl)-2-cyclopentylethanone (INCB38579) is a novel small molecule antagonist of the human and rodent histamine H4 receptors with at least 80-fold selectivity over the human histamine H1, H2 and H3 receptors, and has good pharmacokinetic properties in rats and mice. The compound is potent in inhibiting histamine binding to and signaling through the recombinant human, mouse and rat histamine H4 receptors and blocks the histamine-induced migration of human and mouse dendritic cells, as well as the cell shape change and migration of human eosinophils. INCB38579 and histamine may have separate but overlapping binding sites on the human histamine H4 receptor. This novel inhibitor is efficacious when evaluated in two previously established in vivo models for histamine H4 receptor activity (histamine-induced itch in mice and carrageenan-induced acute inflammatory pain in rats). When examined in formalin-induced pain models, INCB38579 significantly reduces the sustained inflammatory pain experienced by rats and mice. A good correlation between the protein binding adjusted potency from in vitro studies and its analgesic effect in vivo was observed. These results suggest that INCB38579 can serve as a useful tool for pharmacologic characterization of the histamine H4 receptor and further support the hypothesis that targeting the histamine H4 receptor may provide new therapeutic agents for various chronic inflammatory diseases, including inflammatory pain.

Modulation of NMDA receptors by intrathecal administration of the sensory neuron-specific receptor agonist BAM8-22.

The sensory neuron-specific receptor (SNSR) is exclusively distributed in dorsal root ganglion (DRG) cells. We have demonstrated that intrathecal (i.t.) administration of SNSR agonists inhibits formalin-evoked responses and the development of morphine tolerance [Chen, T., Cai, Q., Hong, Y., 2006. Intrathecal sensory neuron-specific receptor agonists bovine adrenal medulla 8-22 and (tyr(6))-gamma2-msh-6-12 inhibit formalin-evoked nociception and neuronal fos-like immunoreactivity in the spinal cord of the rat. Neuroscience 141, 965-975]. The present study was undertaken to examine the possible impact of the activation of SNSR on NMDA receptors. I.t. administration of NMDA (6.8 nmol) induced nociceptive behaviors, including scratching, biting and lifting, followed by thermal hypoalgesia and hyperalgesia. These responses were associated with the expression of Fos-like immunoreactivity (FLI) throughout the spinal dorsal horn with highest effect seen in laminae I-II. I.t. NMDA also induced an increase in nitric oxide synthase (NOS) activity in superficial layers of the dorsal horn, but not around the central canal, as revealed by NADPH diaphorase histochemistry. Pretreatment with the SNSR agonist bovine adrenal medulla 8-22 (3, 10 and 30 nmol) dose-dependently diminished NMDA-evoked nocifensive behaviors and hyperalgesia. This agonist also reduced NMDA-evoked expression of FLI and NADPH reactivity in the spinal dorsal horn. Taken together, these data suggest that the activation of SNSR induces spinal analgesia by suppressing NMDA receptor-mediated activation of spinal dorsal horn neurons and an increase in NOS activity.