Effects of Kv1.3 knockout on pyramidal neuron excitability and synaptic plasticity in piriform cortex of mice.

Kv1.3 belongs to the voltage-gated potassium (Kv) channel family, which is widely expressed in the central nervous system and associated with a variety of neuropsychiatric disorders. Kv1.3 is highly expressed in the olfactory bulb and piriform cortex and involved in the process of odor perception and nutrient metabolism in animals. Previous studies have explored the function of Kv1.3 in olfactory bulb, while the role of Kv1.3 in piriform cortex was less known. In this study, we investigated the neuronal changes of piriform cortex and feeding behavior after smell stimulation, thus revealing a link between the olfactory sensation and body weight in Kv1.3 KO mice. Coronal slices including the anterior piriform cortex were prepared, whole-cell recording and Ca2+ imaging of pyramidal neurons were conducted. We showed that the firing frequency evoked by depolarization pulses and Ca2+ influx evoked by high K+ solution were significantly increased in pyramidal neurons of Kv1.3 knockout (KO) mice compared to WT mice. Western blotting and immunofluorescence analyses revealed that the downstream signaling molecules CaMKII and PKCα were activated in piriform cortex of Kv1.3 KO mice. Pyramidal neurons in Kv1.3 KO mice exhibited significantly reduced paired-pulse ratio and increased presynaptic Cav2.1 expression, proving that the presynaptic vesicle release might be elevated by Ca2+ influx. Using Golgi staining, we found significantly increased dendritic spine density of pyramidal neurons in Kv1.3 KO mice, supporting the stronger postsynaptic responses in these neurons. In olfactory recognition and feeding behavior tests, we showed that Kv1.3 conditional knockout or cannula injection of 5-(4-phenoxybutoxy) psoralen, a Kv1.3 channel blocker, in piriform cortex both elevated the olfactory recognition index and altered the feeding behavior in mice. In summary, Kv1.3 is a key molecule in regulating neuronal activity of the piriform cortex, which may lay a foundation for the treatment of diseases related to piriform cortex and olfactory detection.

Mechanosensitive TRPV4 is required for crystal-induced inflammation.

Crystal structures activate innate immune cells, especially macrophages and initiate inflammatory responses. We aimed to understand the role of the mechanosensitive TRPV4 channel in crystal-induced inflammation. Real-time RT-PCR, RNAscope in situ hybridisation, and Trpv4eGFP mice were used to examine TRPV4 expression and whole-cell patch-clamp recording and live-cell Ca2+ imaging were used to study TRPV4 function in mouse synovial macrophages and human peripheral blood mononuclear cells (PBMCs). Both genetic deletion and pharmacological inhibition approaches were used to investigate the role of TRPV4 in NLRP3 inflammasome activation induced by diverse crystals in vitro and in mouse models of crystal-induced pain and inflammation in vivo. TRPV4 was functionally expressed by synovial macrophages and human PBMCs and TRPV4 expression was upregulated by stimulation with monosodium urate (MSU) crystals and in human PBMCs from patients with acute gout flares. MSU crystal-induced gouty arthritis were significantly reduced by either genetic ablation or pharmacological inhibition of TRPV4 function. Mechanistically, TRPV4 mediated the activation of NLRP3 inflammasome by diverse crystalline materials but not non-crystalline NLRP3 inflammasome activators, driving the production of inflammatory cytokine interleukin-1ß which elicited TRPV4-dependent inflammatory responses in vivo. Moreover, chemical ablation of the TRPV1-expressing nociceptors significantly attenuated the MSU crystal-induced gouty arthritis. In conclusion, TRPV4 is a common mediator of inflammatory responses induced by diverse crystals through NLRP3 inflammasome activation in macrophages. TRPV4-expressing resident macrophages are critically involved in MSU crystal-induced gouty arthritis. A neuroimmune interaction between the TRPV1-expressing nociceptors and the TRPV4-expressing synovial macrophages contributes to the generation of acute gout flares.

Hepatoprotective effect of Herpetospermum caudigerum Wall. on carbon tetrachloride-induced hepatic fibrosis in rats.

Tibetan medicine Herpetospermum caudigerum Wall. (HCW) has long been employed to treat hepatitis, inflammatory diseases and jaundice according to the records of "The Four Medical Tantras" in China. This study was investigated to explore the protective effects of HCW on hepatic fibrosis and the possible mechanism in a rat model. Hepatic fibrosis was established by intragastric administration of 3 ml/kg carbon tetrachloride (CCl4 ) twice a week for 6 weeks. CCl4 -treated rats were received HCW (1 and 3 g/kg/d) and silymarin (0.1 g/kg/d) from 3 to 6 weeks. The results showed that HCW could significantly decrease the levels of AST, ALT, HA, LN, PCIII, Col IV, TNF-α, IL-1ß and IL-6. Moreover, HCW could effectively inhibit collagen deposition and reduce the pathological damage. Analysis experiments finally exhibited that HCW was able to markedly inhibit hepatic fibrosis by modulating the expressions of NF-κB p65, IκBα, Samd3 and TGF-ß1 proteins. Therefore, our results suggest that HCW has hepatoprotective activity against CCl4 -induced hepatic fibrosis in rats by regulating the inflammatory responses.

Salidroside suppressing LPS-induced myocardial injury by inhibiting ROS-mediated PI3K/Akt/mTOR pathway in vitro and in vivo.

The purpose of the present study was to investigate the effect of salidroside (Sal) on myocardial injury in lipopolysaccharide (LPS)-induced endotoxemic in vitro and in vivo. SD rats were randomly divided into five groups: control group, LPS group (15 mg/kg), LPS plus dexamethasone (2 mg/kg), LPS plus Sal groups with different Sal doses (20, 40 mg/kg). Hemodynamic measurement and haematoxylin and eosin staining were performed. Serum levels of creatine kinase (CK), lactate dehydrogenase, the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-px), glutathione, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) were measured after the rats were killed. iNOS, COX-2, NF-κB and PI3K/Akt/mTOR pathway proteins were detected by Western blot. In vitro, we evaluated the protective effect of Sal on rat embryonic heart-derived myogenic cell line H9c2 induced by LPS. Reactive oxygen species (ROS) in H9c2 cells was measured by flow cytometry, and the activities of the antioxidant enzymes CAT, SOD, GSH-px, glutathione-S-transferase, TNF-α, IL-6 and IL-1ß in cellular supernatant were measured. PI3K/Akt/mTOR signalling was examined by Western blot. As a result, Sal significantly attenuated the above indices. In addition, Sal exerts pronounced cardioprotective effect in rats subjected to LPS possibly through inhibiting the iNOS, COX-2, NF-κB and PI3K/Akt/mTOR pathway in vivo. Furthermore, the pharmacological effect of Sal associated with the ROS-mediated PI3K/Akt/mTOR pathway was proved by the use of ROS scavenger, N-acetyl-l-cysteine, in LPS-stimulated H9C2 cells. Our results indicated that Sal could be a potential therapeutic agent for the treatment of cardiovascular disease.

Role of Sinomenine on Complete Freund's Adjuvant-Induced Arthritis in Rats.

The investigation was undertaken to evaluate the effect of sinomenine (Sin) on experimental adjuvant arthritis rats stimulated by Freund's complete adjuvant and explore the corresponding potential molecular mechanism. The content of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6 were detected. Besides, canonical nuclear factor kappa B (NF-κB) pathway was also assessed to evaluate the antiarthritic potential of sinomenine. Pathological sections of rat paws showed sinomenine and diclofenac sodium significantly alleviated articular cartilage lesion, cellular infiltration, epithelial cell degeneration, synovial tissue vasodilation and congestion. The phosphorylations of inhibitor of kappaB alpha and NF-κB subunit p65 were downregulated with the treatment of sinomenine in dose dependent manners, as well as proinflammatory cytokines. Therefore, it was assumed that sinomenine might be a new therapeutic candidate to treat arthritis. © 2016 IUBMB Life, 68(6):429-435, 2016.

Comparison of the efficacy and safety of benzbromarone and febuxostat in gout and hyperuricemia: a systematic review and meta-analysis.

OBJECTIVE: Urate-lowering therapy (ULT) is widely recognized as the primary treatment for hyperuricemia and gout. Xanthine oxidase inhibitors (XOI), particularly febuxostat, have gained popularity as a frontline approach. However, the divergent efficacy and safety between febuxostat and the traditional ULT drug, benzbromarone, remain poorly understood. This knowledge gap necessitates a comprehensive analysis and evidence update to guide drug selection for physicians and patients. METHOD: We conducted a systematic analysis by extracting relevant clinical studies from four medical literature databases. Forest plots, funnel plots, sensitivity analysis, Egger's test, and subgroup analysis were utilized to compare relevant indicators. RESULTS: The advantages and disadvantages of the two drugs were evaluated based on various indicators such as serum uric acid (SUA), triglyceride (TG), urinary uric acid (UUA), white blood cell count (WBC), total cholesterol (TC), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), estimated glomerular filtration rate (eGFR), and serum creatinine (SC). Benzbromarone demonstrated better efficacy in rapidly reducing SUA levels and inhibiting inflammation for hyperuricemia and gout patients. Febuxostat was slightly less effective in lowering SUA, but there was no significant difference in its impact on liver and kidney function after long-term use. CONCLUSION: This study highlights the superiority of benzbromarone in rapidly reducing SUA and inhibiting inflammation. Febuxostat shows comparable effects on liver and kidney function after long-term use. These findings provide valuable insights for clinicians and patients in drug selection. Key Points • Benzbromarone stands out as a highly effective treatment for hyperuricemia and gout, offering rapid reduction of serum uric acid levels and potent anti-inflammatory effects. • When it comes to long-term use, febuxostat demonstrates comparable effects on liver and kidney function. This provides reassurance for patients who require extended treatment duration. • Moreover, our study goes beyond previous research by presenting a more comprehensive and detailed analysis.

Piper longum L. ameliorates gout through the MAPK/PI3K-AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gout, a painful joint disease with a prevalence ranging from 0.86% to 2.2% in China over the past decade. Traditional medicine has long utilized the medicinal and edible Piper longum L. (PL) fruit spikes for treating gout and other joint conditions like rheumatoid arthritis. However, the exact mechanisms behind its effectiveness remain unclear. AIM OF THE STUDY: This study aimed to investigate the potential of alcoholic extracts from PL fruit spikes as a safe and effective treatment for gout. We used a combined network pharmacology and experimental validation approach to evaluate the mechanisms behind the anti-gout properties of PL. MATERIALS AND METHODS: UPLC-Q/TOF-MS analysis determined the major components of PL. Subsequently, network pharmacology analysis predicted potential molecular targets and related signaling pathways for the anti-gout activity of PL. Molecular docking simulations further explored the interactions between PL compounds and proteins and characterized the properties of potential bioactive secondary metabolites. Mouse models of air pouch inflammation and hyperuricemia were further established, and the anti-gout mechanism of PL was confirmed by examining the expression of proteins related to the MAPK and PI3K-AKT pathways in the tissue. RESULTS: Our analysis revealed 220 bioactive secondary metabolites within PL extracts. Network pharmacology and molecular docking results indicated that these metabolites primarily combat gout by modulating the PI3K-AKT and MAPK signaling pathways. In vivo experiments have also proven that PL at a dose of 100 mg/kg can optimally reduce acute inflammation of gout and kidney damage caused by high uric acid. The anti-gout mechanism involves the PI3K-AKT/MAPK signaling pathway and its downstream NF-κB pathway. CONCLUSION: This study provides compelling evidence for PL's therapeutic potential in gout management by modulating key inflammatory pathways. The findings offer a strong foundation for future clinical exploration of PL as a gout treatment option.

Chrysanthemum morifolium attenuates metabolic and alcohol-associated liver disease via gut microbiota and PPARα/γ activation.

BACKGROUND: Metabolic and alcohol-associated liver disease (MetALD) shows a high prevalence rate in liver patients, but there is currently no effective treatment for MetALD. As a typical edible traditional Chinese medicinal herb, the anti-inflammatory, antioxidant, and hepatoprotective properties of water extract of Chrysanthemum morifolium Ramat. (WECM) has been demonstrated. However, its therapeutic effect on MetALD and the associated mechanisms remain unclear. PURPOSE: To investigate the underlying mechanisms of WECM against MetALD. METHODS: We constructed a MetALD rat model following a high-fat & high-sucrose plus alcohol diet (HFHSAD). MetALD rats were treated with WECM at 2.1, 4.2, and 8.4 g/kg/d for six weeks. Efficacy was determined, and pathways associated with WECM against MetALD were predicted through serum and hepatic biochemical marker measurement, histopathological section analysis, 16S rDNA sequencing of the gut microbiota and untargeted serum metabolomics analyses. Changes in genes and proteins in the peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ) signaling pathways were detected by RT‒PCR and Western blotting. RESULTS: WECM treatment significantly attenuated hepatic steatosis, hyperlipidemia and markers of liver injury in MetALD rats. Moreover, WECM improved vascular endothelial function, hypertension, and systematic oxidative stress. Mechanistically, WECM treatment altered the overall structure of the gut microbiota through maintaining Firmicutes/Bacteroidota ratio and reducing harmful bacterial abundances such as Clostridium, Faecalibaculum, and Herminiimonas. Notably, WECM promoted 15-deoxy-△12, 14-prostaglandin J2 (15d-PGJ2) release and further activated the PPARγ to reduce serum TNF-α, IL-1ß, and IL-6 levels. Additionally, WECM upregulated PPARα and downregulated the levels of CD36 and FABP4 to improve lipid metabolism. CONCLUSION: Our findings provide the first evidence that WECM treatment significantly improved hepatic steatosis, oxidative stress and inflammation in MetALD rats by regulating the gut microbiota and activating the 15d-PGJ2/PPARγ and PPARα signaling pathway.

Attenuation of gouty arthritis by emodinol in monosodium urate crystal-treated mice.

A series of studies have recently demonstrated that the release of interleukin 1ß induced by monosodium urate crystals is central to the experimental gouty arthritis. Elaeagnus pungens has been traditionally used for the treatment of gouty arthritis in China for more than thousands years. However, there is still little known about the active ingredients and mechanisms of E. pungens against gouty arthritis. Emodinol, as a major triterpene compound in E. pungens, has been seldom reported to have an effect on gouty arthritis. Therefore, the potential beneficial effects and mechanisms of emodinol on gouty arthritis were investigated in this study. Results showed that it significantly ameliorated the hyperalgesia, inflammation, and levels of multiple proinflammatory cytokines in monosodium urate crystals-treated mice. These findings elucidate that emodinol exhibits a prominent effect on improving symptoms of acute gouty arthritis induced by monosodium urate crystals through inhibiting the generation of proinflammatory cytokines.

Oral delivery of porous starch-loaded bilayer microgels for controlled drug delivery and treatment of ulcerative colitis.

We prepared one type of bilayer microgels for oral administration with three effects: pH responsiveness, time lag, and colon enzyme degradation. Combined with the dual biological effects of curcumin (Cur) for reducing inflammation and promoting repair of colonic mucosal injury, targeted colonic localization and release of Cur according to the colonic microenvironment were enhanced. The inner core, derived from guar gum and low-methoxyl pectin, afforded colonic adhesion and degradation behavior; the outer layer, modified by alginate and chitosan via polyelectrolyte interaction, achieved colonic localization. The porous starch (PS)-mediated strong adsorption allowed Cur loading in inner core to achieve a multifunctional delivery system. In vitro, the formulations exhibited good bioresponses at different pH conditions, potentially delaying Cur release in the upper gastrointestinal tract. In vivo, dextran sulfate sodium-induced ulcerative colitis (UC) symptoms were significantly alleviated after oral administration, accompanied by reduced levels of inflammatory factors. The formulations facilitated colonic delivery, allowing Cur accumulation in colonic tissue. Moreover, the formulations could alter gut microbiota composition in mice. During Cur delivery, each formulation increased species richness, decreased pathogenic bacterial content, and afforded synergistic effects against UC. These PS-loaded bilayer microgels, exhibiting excellent biocompatibility, multi-bioresponsiveness, and colon targeting, could be beneficial in UC therapy, allowing development into a novel oral formulation.

Polygoni Multiflori Radix Praeparata and Acori Tatarinowii Rhizoma ameliorate scopolamine-induced cognitive impairment by regulating the cholinergic and synaptic associated proteins.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Polygoni Multiflori Radix Praeparata (PMRP) and Acori Tatarinowii Rhizoma (ATR) is often used in traditional Chinese medicine to prevent and treat Alzheimer's disease (AD). However, it is not clear whether the effects and mechanisms of the decoction prepared by traditional decocting method (PA) is different from that prepared by modern decocting method (P + A). AIM OF THE STUDY: The present study aimed to investigate the differences in the protective effects of PA and P + A on scopolamine induced cognitive impairment, and to explore its potential mechanism. MATERIALS AND METHODS: To assess the protective effect of PA and P + A on cognitive dysfunction, the mice were orally administrated with PA (1.56, 6.24 g kg-1•day-1) and P + A (1.56, 6.24 g kg-1•day-1) for 26 days before co-treatment with scopolamine (4 mg kg-1•day-1, i.p.). The learning and memory abilities of mice were examined by Morris water maze test, and the expressions of proteins related to cholinergic system and synaptic function were detected by the methods of ELISA, real-time PCR and Western blotting. Then, molecular docking technique was used to verify the effect of active compounds in plasma after PA administration on Acetylcholinesterase (AChE) protein. Finally, the Ellman method was used to evaluate the effects of different concentrations of PA, P + A (1 µg/mL-100 mg/mL) and the compounds (1-100 µM) on AChE activity in vitro. RESULTS: On one hand, in the scopolamine-induced cognitive impairment mouse model, both of PA and P + A could improve the cognitive impairment, while the effect of PA on cognitive amelioration was better than that of P + A. Moreover, PA regulated the cholinergic and synaptic functions by enhancing the concentration of acetylcholine (ACh), the mRNA levels of CHT1, Syn, GAP-43 and PSD-95, and the related proteins (CHT1, VACHT, Syn, GAP-43 and PSD-95), and significantly inhibiting the expression of AChE protein. Meanwhile, P + A only up-regulated the mRNA levels of GAP-43 and PSD-95, increased the expressions of CHT1, VACHT, Syn, GAP-43 and PSD-95 proteins, and inhibited the expression of AChE protein. On the other hand, the in vitro study showed that some compounds including emodin-8-o-ß-d-Glucopyranoside, THSG and α-Asarone inhibited AChE protein activity with the IC50 values 3.65 µM, 5.42 µM and 9.43 µM, respectively. CONCLUSIONS: These findings demonstrate that both of PA and P + A can ameliorate the cognitive deficits by enhancing cholinergic and synaptic related proteins, while PA has the stronger improvement effect on the cholinergic function, which may be attributed to the compounds including THSG, emodin, emodin-8-O-ß-D-glucopyranoside and α-asarone. The present study indicated that PA has more therapeutic potential in the treatment of neurodegenerative diseases such as AD. The results provide the experimental basis for the clinical use of PA.

Potential anti-gout properties of Wuwei Shexiang pills based on network pharmacology and pharmacological verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuwei Shexiang Pills (WWSX), a classic Tibetan medicine, consists of Chebulae Fructus (removed pit), Aucklandiae Radix, Moschus, Aconiti Fiavi Radix, and Acori Calami Rhizoma. It is used clinically in China to treat joint pain, swelling and other symptoms, and has the function of dispelling wind and relieving pain. However, to date, the mechanism of how it works against gout is still unclear. AIMS OF THE STUDY: Using network pharmacology, molecular docking and pharmacological verification to explore the potential anti-gout properties of WWSX. MATERIALS AND METHODS: With the use of UPLC-Q/TOF-MS, the main components of WWSX were obtained and screened for potential anti-inflammatory components by network pharmacology and molecular docking. The anti-inflammatory activity of the components screened from WWSX was also tested by in vitro assays. The anti-gout mechanism of WWSX was predicted by network pharmacology, and the pharmacological validation experiments using gouty arthritis model and mouse air pouch model were used to explore the multifaceted mechanism of WWSX to modify gout. RESULT: Thirty-eight active ingredients were obtained from the UPLC-Q/TOF-MS detection. The network pharmacology and molecular docking analysis showed that 104 co-targets were participated in the treatment of gout, and the main signaling pathways involved were NOD-like receptor pathway, NF-κB pathway and MAPK pathway. Pharmacological evaluation showed that WWSX could significantly improve gout in gouty arthritis models and mouse air pouch models by modulating the above pathways. CONCLUSION: This work has predicted and validated the anti-inflammatory material basis and predicted the anti-gout mechanism of WWSX which was verified by network pharmacology, molecular docking and in vitro cellular studies. The results reveal the mechanism of WWSX in the treatment of gout and provide a theoretical basis for its clinical application.

Chemotherapy-induced phlebitis via the GBP5/NLRP3 inflammasome axis and the therapeutic effect of aescin.

BACKGROUND AND PURPOSE: Intravenous infusion of chemotherapy drugs can cause severe chemotherapy-induced phlebitis (CIP) in patients. However, the underlying mechanism of CIP development remains unclear. EXPERIMENTAL APPROACH: RNA-sequencing analysis was used to identify potential disease targets in CIP. Guanylate binding protein-5 (GBP5) genetic deletion approaches also were used to investigate the role of GBP5 in NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in lipopolysaccharide (LPS) primed murine bone-marrow-derived macrophages (BMDMs) induced by vinorelbine (VIN) in vitro and in mouse models of VIN-induced CIP in vivo. The anti-CIP effect of aescin was evaluated, both in vivo and in vivo. KEY RESULTS: Here, we show that the expression of GBP5 was upregulated in human peripheral blood mononuclear cells from CIP patients. Genetic ablation of GBP5 in murine macrophages significantly alleviated VIN-induced CIP in the experimental mouse model. Mechanistically, GBP5 contributed to the inflammatory responses through activating NLRP3 inflammasome and driving the production of the inflammatory cytokine IL-1ß. Moreover, aescin, a mixture of triterpene saponins extracted from horse chestnut seed, can alleviate CIP by inhibiting the GBP5/NLRP3 axis. CONCLUSION AND IMPLICATIONS: These findings suggest that GBP5 is an important regulator of NLRP3 inflammasome in CIP mouse model. Our work further reveals that aescin may serve as a promising candidate in the clinical treatment of CIP.

Piezo2 channels expressed by colon-innervating TRPV1-lineage neurons mediate visceral mechanical hypersensitivity.

Inflammatory and functional gastrointestinal disorders such as irritable bowel syndrome (IBS) and obstructive bowel disorder (OBD) underlie the most prevalent forms of visceral pain. Although visceral pain can be generally provoked by mechanical distension/stretch, the mechanisms that underlie visceral mechanosensitivity in colon-innervating visceral afferents remain elusive. Here, we show that virally mediated ablation of colon-innervating TRPV1-expressing nociceptors markedly reduces colorectal distention (CRD)-evoked visceromotor response (VMR) in mice. Selective ablation of the stretch-activated Piezo2 channels from TRPV1 lineage neurons substantially reduces mechanically evoked visceral afferent action potential firing and CRD-induced VMR under physiological conditions, as well as in mouse models of zymosan-induced IBS and partial colon obstruction (PCO). Collectively, our results demonstrate that mechanosensitive Piezo2 channels expressed by TRPV1-lineage nociceptors powerfully contribute to visceral mechanosensitivity and nociception under physiological conditions and visceral hypersensitivity under pathological conditions in mice, uncovering potential therapeutic targets for the treatment of visceral pain.

The immunosuppressive effects and mechanisms of loureirin B on collagen-induced arthritis in rats.

Introduction: Rheumatoid arthritis (RA) is a common disease mainly affecting joints of the hands and wrists. The discovery of autoantibodies in the serum of patients revealed that RA belonged to the autoimmune diseases and laid a theoretical basis for its immunosuppressive therapy. The pathogenesis of autoimmune diseases mainly involves abnormal activation and proliferation of effector memory T cells, which is closely related to the elevated expression of Kv1.3, a voltage-gated potassium (Kv) channel on the effector memory T cell membrane. Drugs blocking the Kv1.3 channel showed a strong protective effect in RA model animals, suggesting that Kv1.3 is a target for the discovery of specific RA immunosuppressive drugs. Methods: In the present study, we synthesized LrB and studied the effects of LrB on collagen- induced arthritis (CIA) in rats. The clinical score, paw volume and joint morphology of CIA model rats were compared. The percentage of CD3+, CD4+ and CD8+ T cells in rat peripheral blood mononuclear and spleen were analyzed with flow cytometry. The concentrations of inflammatory cytokines interleukin (IL)-1b, IL-2, IL-4, IL-6, IL-10 and IL-17 in the serum of CIA rats were analyzed with enzyme-linked immunosorbent assay. The IL-1b and IL-6 expression in joints and the Kv1.3 expression in peripheral blood mononuclear cells (PBMCs) were quantified by qPCR. To further study the mechanisms of immunosuppressive effects of LrB, western blot and immunofluorescence were utilized to study the expression of Kv1.3 and Nuclear Factor of Activated T Cells 1 (NFAT1) in two cell models - Jurkat T cell line and extracted PBMCs. Results: LrB effectively reduced the clinical score and relieved joint swelling. LrB could also decrease the percentage of CD4+ T cells, while increase the percentage of CD8+ T cells in peripheral blood mononuclear and spleen of rats with CIA. The concentrations of inflammatory cytokines interleukin (IL)-1b, IL-2, IL-6, IL-10 and IL-17 in the serum of CIA rats were significantly reduced by LrB. The results of qPCR showed that Kv1.3 mRNA in the PBMCs of CIA rats was significantly higher than that of the control and significantly decreased in the LrB treatment groups. In addition, we confirmed in cell models that LrB significantly decreased Kv1.3 protein on the cell membrane and inhibited the activation of Nuclear Factor of Activated T Cells 1 (NFAT1) with immune stimulus. Conclusion: In summary, this study revealed that LrB could block NFAT1 activation and reduce Kv1.3 expression in activated T cells, thus inhibiting the proliferation of lymphocytes and the release of inflammatory cytokines, thereby effectively weakening the autoimmune responses in CIA rats. The effects of immunosuppression due to LrB revealed its potential medicinal value in the treatment of RA.

A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain.

Microglia, resident macrophages of the CNS, are essential to brain development, homeostasis, and disease. Microglial activation and proliferation are hallmarks of many CNS diseases, including neuropathic pain. However, molecular mechanisms that govern the spinal neuroimmune axis in the setting of neuropathic pain remain incompletely understood. Here, we show that genetic ablation or pharmacological blockade of transient receptor potential vanilloid type 4 (TRPV4) markedly attenuated neuropathic pain-like behaviors in a mouse model of spared nerve injury. Mechanistically, microglia-expressed TRPV4 mediated microglial activation and proliferation and promoted functional and structural plasticity of excitatory spinal neurons through release of lipocalin-2. Our results suggest that microglial TRPV4 channels reside at the center of the neuroimmune axis in the spinal cord, which transforms peripheral nerve injury into central sensitization and neuropathic pain, thereby identifying TRPV4 as a potential new target for the treatment of chronic pain.

Epimedii Folium and Curculiginis Rhizoma ameliorate lipopolysaccharides-induced cognitive impairment by regulating the TREM2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Neuroinflammation induced by microglia is closely related to a variety of neurodegenerative diseases including Alzheimer's disease (AD). Previous study has found that aqueous extract of Epimedii Folium and Curculiginis Rhizoma (EX) had anti-inflammatory effect on AD by activating the NLRP3 inflammasome and inhibiting NF-κB/MAPK pathway. However, whether the anti-neuroinflammatory effect of EX is related to microglia or not remains unclear. AIM OF THE STUDY: The present study aimed to investigate the protective effect of EX on cognitive impairment induced by LPS and explore the underlying mechanism of EX. MATERIALS AND METHODS: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was performed to qualify the major components of EX, EX in the serum and cerebrospinal fluid. To evaluate the anti-inflammatory effects of EX in vivo, the mice were orally administrated with EX (2.34, 4.68 g kg-1•d-1) for 28 days before cotreatment with LPS (1 mg kg-1•d-1, i.p.). The leaning and memory abilities of mice were examined by Morris water maze test. The expression of inflammatory related proteins and the activation of microglia were detected by ELISA, immunofluorescence, real-time PCR and Western blotting. RESULTS: HPLC-MS analysis confirmed and quantified 9 components in EX, 5 components in the serum and 4 components in the cerebrospinal fluid. In a LPS-induced neuroinflammatory mouse model, EX was found to exert anti-inflammatory activity by reducing the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), regulating the expression of different phenotypes of microglia, and increasing the expression of proteins related with TREM2 in the hippocampus tissue. Moreover, LPS-induced microglia activation was markedly attenuated in the hippocampus. CONCLUSIONS: These findings demonstrate that EX exerts anti-neuroinflammatory effects via reducing the production of inflammatory mediators, regulating the conversion of microglia and activating the proteins related with TREM2. EX might become a novel herb pairs to treat neuroinflammatory diseases.

Integrating network pharmacology and pharmacological validation to explore the effect of Shi Wei Ru Xiang powder on suppressing hyperuricemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Shi Wei Ru Xiang powder (SWR) is a traditional Tibetan medicinal formula with the effect of dispelling dampness and dispersing cold. In clinical practice, SWR is generally used for the treatment of hyperuricemia (HUA). However, its exact pharmacological mechanism remains unclear. AIMS OF THE STUDY: To preliminarily elucidate the regulatory effects and possible mechanisms of SWR on hyperuricemia using network pharmacology and experimental validation. MATERIALS AND METHODS: A mouse model of hyperuricemia was used to evaluate the alleviating effect of SWR on hyperuricemia. The major components of SWR were acquired by UPLC-Q/TOF-MS. The potential molecular targets and associated signaling pathways were predicted through network pharmacology. The mechanism of action of SWR in ameliorating hyperuricemia was further investigated by pharmacological evaluation. RESULTS: Mice with hyperuricemia and renal dysfunction were ameliorated by SWR. The 36 components of SWR included phenolic acids, terpenoids, alkaloids and flavonoids were identified. Network pharmacological analysis showed the involvement of the above compounds, and 115 targets were involved to treat hyperuricemia, involving multiple biological processes and different signaling pathways. Pharmacological experiments validated that SWR ameliorated hyperuricemic nephropathy in mice by modulating the mitogen-activated protein kinase (MAPK) signaling pathway, nuclear factor kappaB (NF-κB) signaling pathway and NOD-like receptor signaling pathway. CONCLUSION: MAPK signaling pathway, NF-κB signaling pathway and NOD-like receptor signaling pathway play important roles in the therapeutic effects of SWR on hyperuricemia.

Astilbin attenuates hyperuricemia and ameliorates nephropathy in fructose-induced hyperuricemic rats.

Astilbin is a flavonoid compound isolated from the rhizome of Smilax china L. The effects and possible mechanisms of astilbin on hyperuricemia and nephropathy rats were elucidated in this study. Different dosages of astilbin (1.25, 2.5, and 5.0 mg/kg) were administered to 10 % fructose-induced hyperuricemic rats. The results demonstrated that astilbin significantly decreased the serum uric acid (Sur) level by increasing the urinary uric acid (Uur) level and fractional excretion of urate (FEUA) but not inhibiting the xanthine oxidase (XOD) activity. In addition, kidney function parameters such as serum creatinine (Scr) and blood urea nitrogen (BUN) were recovered in astilbin-treated hyperuricemic rats. Further investigation indicated that astilbin prevented the renal damage against the expression of transforming growth factor- ß1 (TGF-ß1) and connective tissue growth factor (CTGF) and also exerted a renal protective role by inhibiting formation of monosodium urate (MSU) and production of prostaglandin E2 (PGE2) and interleukin-1 (IL-1). These findings provide potent evidence for astilbin as a safe and promising lead compound in the development of a disease-modifying drug to prevent hyperuricemia and nephropathy.

A triterpenoid from Thalictrum fortunei induces apoptosis in BEL-7402 cells through the P53-induced apoptosis pathway.

Thalictrum fortunei S. Moore, a perennial plant distributed in the southeastern part of China, has been used in Traditional Chinese Medicine for thousands of years for its antitumor, antibacterial and immunoregulatory effects. In order to investigate the active components and the mechanism of the anti-tumor effects of Thalictrum fortunei, the growth inhibitory effects of eight triterpenoids isolated from the aerial parts of the plant on tumor cell lines were examined by 3-(4,5)-dimethylthiazoy1-3,5-diphenyltetrazolium bromide (MTT) assay. The MTT-assay results showed that the inhibitory activity of 3-O-ß-D-glucopyranosyl-(1→4)-ß-D-fucopyranosyl(22S,24Z)-cycloart-24-en-3ß,22,26-triol 26-O-ß-D-glucopyranoside (1) was stronger than that of the other seven tested triterpenoids on human hepatoma Bel-7402 cell line (Bel-7402), human colon lovo cells (LoVo), human non-small cells lung cancer NCIH-460 cells (NCIH-460) and human gastric carcinoma SGC-7901 cells (SGC-7901) after 48 h treatment in vitro, with the IC(50) values of 66.4, 84.8, 73.5, 89.6 µM, respectively. Moreover, the antitumor mechanism of compound 1 on Bel-7402 cell was explored through nucleus dyeing, fluorescence assay, flow cytometry and western blot. The flow cytometric analysis results revealed that compound 1 caused apoptosis and mitochondrial membrane potential (MMP) loss in Bel-7402 cells. A fluorescence assay indicated that intracellular reactive oxygen species (ROS) were markedly provoked by compound 1 treatment compared to control cells. Immunoblot results showed that compound 1 significantly increased the expression levels of cleaved caspase-3, P53 and Bax protein, and decreased the expression level of Bcl-2 protein. These findings indicate that compound 1 inhibits the growth activity of tumor cells, probably through the P53 protein-induced apoptosis pathway.