Discovery of a novel anti-osteoporotic agent from marine fungus-derived structurally diverse sirenins.

Thirteen new sirenin derivatives named eupenicisirenins C-O (1-13), along with a biosynthetically related known one (14), were isolated from the mangrove sediment-derived fungus Penicillium sp. SCSIO 41410. The structures, which possessed a rare cyclopropane moiety, were confirmed by extensive analyses of the spectroscopic data, quantum chemical calculations, and X-ray diffraction. Among them, eupenicisirenin C (1) exhibited the strongest NF-κB inhibitory activities, as well as suppressing effects on cGAS-STING pathway. Moreover, 1 showed the significant inhibitory effect on RANKL-induced osteoclast differentiation in bone marrow macrophages cells, and also displayed the therapeutic potential on prednisolone-induced zebrafish osteoporosis. Transcriptome analysis and the following verification tests suggested that its anti-osteoporotic mechanism is related to the extracellular matrix receptor interaction-related pathways. This study provided a promising marine-derived anti-osteoporotic agent for the treatment of skeletal disease.

The volume regulated anion channel VRAC regulates NLRP3 inflammasome by modulating itaconate efflux and mitochondria function.

The NLRP3 inflammasome is a supramolecular complex that is linked to sterile and pathogen-dependent inflammation, and its excessive activation underlies many diseases. Ion flux disturbance and cell volume regulation are both reported to mediate NLRP3 inflammasome activation, but the underlying orchestrating signaling remains not fully elucidated. The volume-regulated anion channel (VRAC), formed by LRRC8 proteins, is an important constituent that controls cell volume by permeating chloride and organic osmolytes in response to cell swelling. We now demonstrate that Lrrc8a, the essential component of VRAC, plays a central and specific role in canonical NLRP3 inflammasome activation. Moreover, VRAC acts downstream of K+ efflux for NLRP3 stimuli that require K+ efflux. Mechanically, our data demonstrate that VRAC modulates itaconate efflux and damaged mitochondria production for NLRP3 inflammasome activation. Further in vivo experiments show mice with Lrrc8a deficiency in myeloid cells were protected from lipopolysaccharides (LPS)-induced endotoxic shock. Taken together, this work identifies VRAC as a key regulator of NLRP3 inflammasome and innate immunity by regulating mitochondrial adaption for macrophage activation and highlights VRAC as a prospective drug target for the treatment of NLRP3 inflammasome and itaconate related diseases.

A novel TWIK2 channel inhibitor binds at the bottom of the selectivity filter and protects against LPS-induced experimental endotoxemia in vivo.

TWIK2 channel plays a critical role in NLRP3 inflammasome activation and mice deficient in TWIK2 channel are protected from sepsis and inflammatory lung injury. However, inhibitors of TWIK2 channel are currently in an early stage of development, and the molecular determinants underlying the chemical modulation of TWIK2 channel remain unexplored. In this study, we identified NPBA and the synthesized derivative NPBA-4 potently and selectively inhibited TWIK2 channel by using whole-cell patch clamp techniques. Furthermore, the mutation of the last residues of the selectivity filter in both P1 and P2 (i.e., T106A, T214A) of TWIK2 channel substantially abolished the effect of NPBA on TWIK2 channel. Our data suggest that NPBA blocked TWIK2 channel through binding at the bottom of the selectivity filter, which was also supported by molecular docking prediction. Moreover, we found that NPBA significantly suppressed NLRP3 inflammasome activation in macrophages and alleviated LPS-induced endotoxemia and organ injury in vivo. Notably, the protective effects of NPBA against LPS-induced endotoxemia were abolished in Kcnk6-/- mice. In summary, our study has uncovered a series of novel inhibitors of TWIK2 channel and revealed their distinct molecular determinants interacting TWIK2 channel. These findings provide new insights into the mechanisms of pharmacological action on TWIK2 channel and opportunities for the development of selective TWIK2 channel modulators to treat related inflammatory diseases.

TRPV4 channel is involved in HSV-2 infection in human vaginal epithelial cells through triggering Ca2+ oscillation.

Herpes simplex virus (HSV) infection induces a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), which plays a critical role in facilitating viral entry. T-type calcium channel blockers and EGTA, a chelate of extracellular Ca2+, suppress HSV-2 infection. But the cellular mechanisms mediating HSV infection-activated Ca2+ signaling have not been completely defined. In this study we investigated whether the TRPV4 channel was involved in HSV-2 infection in human vaginal epithelial cells. We showed that the TRPV4 channel was expressed in human vaginal epithelial cells (VK2/E6E7). Using distinct pharmacological tools, we demonstrated that activation of the TRPV4 channel induced Ca2+ influx, and the TRPV4 channel worked as a Ca2+-permeable channel in VK2/E6E7 cells. We detected a direct interaction between the TRPV4 channel protein and HSV-2 glycoprotein D in the plasma membrane of VK2/E6E7 cells and the vaginal tissues of HSV-2-infected mice as well as in phallic biopsies from genital herpes patients. Pretreatment with specific TRPV4 channel inhibitors, GSK2193874 (1-4 µM) and HC067047 (100 nM), or gene silence of the TRPV4 channel not only suppressed HSV-2 infectivity but also reduced HSV-2-induced cytokine and chemokine generation in VK2/E6E7 cells by blocking Ca2+ influx through TRPV4 channel. These results reveal that the TRPV4 channel works as a Ca2+-permeable channel to facilitate HSV-2 infection in host epithelial cells and suggest that the design and development of novel TRPV4 channel inhibitors may help to treat HSV-2 infections.

Novel DCPIB analogs as dual inhibitors of VRAC/TREK1 channels reduced cGAS-STING mediated interferon responses.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA and catalyzes the formation of 2'3'-cyclic-GMP-AMP (cGAMP), which in turn triggers interferon (IFN) production. Inappropriate activation of cGAS and production of cGAMP have been linked to a diversity of autoimmune diseases. The volume-regulated anion channels (VRACs) have been recently demonstrated to permeate cGAMP, thus making the channel essential for the activation of the cGAS-cGAMP-STING axis. DCPIB, a prominent inhibitor of VRAC channel, has been recently reported to also significantly activate TREK1 channel. Herein, in this study, we have designed and synthesized a series of novel DCPIB derivatives and investigated their potential regulatory effects on VRAC/TREK1 channels. Our results manifested that compound 6u was a dual inhibitor of VRAC/TREK1 channels with IC50s of 7.11 ± 0.94 µM and 4.43 ± 0.90 µM, respectively. On top of that, our data demonstrated that 6u impaired interferon production in a concentration-dependently manner by dampening cGAS-cGAMP-STING pathway without any cytotoxicity when it comes to herpes simplex virus type 1 (HSV1) infection. To sum up, our study not only discovered a novel DCPIB analog with dual inhibitory effects on VRAC/TREK1 channels but also provided a new strategy for the design and development of newly potent VRAC inhibitors, which benefits the treatment of cGAS-STING related autoimmune and inflammatory diseases.

Pharmacological inhibition of Kv1.3 channel impairs TLR3/4 activation and type I IFN response and confers protection against Listeria monocytogenes infection.

Emerging data have demonstrated the critical roles of potassium efflux in the innate immune system. However, the role of potassium efflux in TLR3/4 activation and type I interferon (IFN) responses are not well elucidated. In the present study, we found potassium efflux is essential for TLR3/4 signaling, which mediates the expression of IFN and its inducible gene Cxcl10 and proinflammatory cytokine gene TNF-α. Furthermore, pharmacological inhibition of Kv1.3 channel (PAP-1), but not Kir2.1, KCa3.1 or TWIK2, attenuated TLR3/4 receptor activation in macrophages. Mechanistically, PAP-1 suppressed LPS-induced inflammatory function through marked suppressing the activation of JNK mitogen-activated protein kinase (MAPK) and p65 subunit of nuclear factor-kB (NF-kB). Notably, PAP-1 effectively protected mice against Listeria monocytogenes induced infection. Our findings reveal that potassium efflux mediated by the Kv1.3 channel is essential for TLR3/4 activation and suggest that pharmacological inhibition of Kv1.3 may help to treat type I IFN related autoimmune diseases and bacterial infections.

Total Ginsenoside Extract from Panax ginseng Enhances Neural Stem Cell Proliferation and Neuronal Differentiation by Inactivating GSK-3ß.

OBJECTIVE: To study the effects of total ginsenosides (TG) extract from Panax ginseng on neural stem cell (NSC) proliferation and differentiation and their underlying mechanisms. METHODS: The migration of NSCs after treatment with various concentrations of TG extract (50, 100, or 200 µ g/mL) were monitored. The proliferation of NSCs was examined by a combination of cell counting kit-8 and neurosphere assays. NSC differentiation mediated by TG extract was evaluated by Western blotting and immunofluorescence staining to monitor the expression of nestin and microtubule associated protein 2 (MAP2). The GSK-3ß/ß-catenin pathway in TG-treated NSCs was examined by Western blot assay. The NSCs with constitutively active GSK-3ß mutant were made by adenovirus-mediated gene transfection, then the proliferation and differentiation of NSCs mediated by TG were further verified. RESULTS: TG treatment significantly enhanced NSC migration (P<0.01 or P<0.05) and increased the proliferation of NSCs (P<0.01 or P<0.05). TG mediation also significantly upregulated MAP2 expression but downregulated nestin expression (P<0.01 or P<0.05). TG extract also significantly induced GSK-3ß phosphorylation at Ser9, leading to GSK-3ß inactivation and, consequently, the activation of the GSK-3ß/ß-catenin pathway (P<0.01 or P<0.05). In addition, constitutive activation of GSK-3ß in NSCs by the transfection of GSK-3ß S9A mutant was found to significantly suppress TG-mediated NSC proliferation and differentiation (P<0.01 or P<0.05). CONCLUSION: TG promoted NSC proliferation and neuronal differentiation by inactivating GSK-3ß.

Discovery of novel verinurad analogs as dual inhibitors of URAT1 and GLUT9 with improved Druggability for the treatment of hyperuricemia.

Verinurad (RDEA3170) is a selective URAT1 inhibitor under investigation for the treatment of gout and hyperuricemia. In an effort to further improve the pharmacodynamics/pharmacokinetics of verinurad and to increase the structural diversity, we designed novel verinurad analogs by introducing a linker (e.g. aminomethyl, amino or oxygen) between the naphthalene and the pyridine ring to increase the flexibility. These compounds were synthesized and tested for their in vitro URAT1-inhibitory activity. Most compounds exhibited potent inhibitory activities against URAT1 with IC50 values ranging from 0.24 µM to 16.35 µM. Among them, compound KPH2f exhibited the highest URAT1-inhibitory activity with IC50 of 0.24 µM, comparable to that of verinurad (IC50 = 0.17 µM). KPH2f also inhibited GLUT9 with an IC50 value of 9.37 ± 7.10 µM, indicating the dual URAT1/GLUT9 targeting capability. In addition, KPH2f showed little effects on OAT1 and ABCG2, and thus was unlikely to cause OAT1/ABCG2-mediated drug-drug interactions and/or to neutralize the uricosuric effects of URAT1/GLUT9 inhibitors. Importantly, KPH2f (10 mg/kg) was equally effective in reducing serum uric acid levels and exhibited higher uricosuric effects in a mice hyperuricemia model, as compared to verinurad (10 mg/kg). Furthermore, KPH2f demonstrated favorable pharmacokinetic properties with an oral bioavailability of 30.13%, clearly better than that of verinurad (21.47%). Moreover, KPH2f presented benign safety profiles without causing hERG toxicity, cytotoxicity in vitro (lower than verinurad), and renal damage in vivo. Collectively, these results suggest that KPH2f represents a novel, safe and effective dual URAT1/GLUT9 inhibitor with improved druggabilities and is worthy of further investigation as an anti-hyperuricemic drug candidate.

ML365 inhibits TWIK2 channel to block ATP-induced NLRP3 inflammasome.

Dysregulation of NLRP3 inflammasome results in uncontrolled inflammation, which participates in various chronic diseases. TWIK2 potassium channel mediates potassium efflux that has been reported to be an essential upstream mechanism for ATP-induced NLRP3 inflammasome activation. Thus, TWIK2 potassium channel could be a potential drug target for NLRP3-related inflammatory diseases. In the present study we investigated the effects of known K2P channel modulators on TWIK2 channel expressed in a heterologous system. In order to increase plasma membrane expression and thus TWIK2 currents, a mutant channel with three mutations (TWIK2I289A/L290A/Y308A) in the C-terminus was expressed in COS-7 cells. TWIK2 currents were assessed using whole-cell voltage-clamp recording. Among 6 known K2P channel modulators tested (DCPIB, quinine, fluoxetine, ML365, ML335, and TKDC), ML365 was the most potent TWIK2 channel blocker with an IC50 value of 4.07 ± 1.5 µM. Furthermore, ML365 selectively inhibited TWIK2 without affecting TWIK1 or THIK1 channels. We showed that ML365 (1, 5 µM) concentration-dependently inhibited ATP-induced NLRP3 inflammasome activation in LPS-primed murine BMDMs, whereas it did not affect nigericin-induced NLRP3, or non-canonical, AIM2 and NLRC4 inflammasomes activation. Knockdown of TWIK2 significantly impaired the inhibitory effect of ML365 on ATP-induced NLRP3 inflammasome activation. Moreover, we demonstrated that pre-administration of ML365 (1, 10, 25 mg/kg, ip) dose-dependently ameliorated LPS-induced endotoxic shock in mice. In a preliminary pharmacokinetic study conducted in rats, ML365 showed good absolute oral bioavailability with F value of 22.49%. In conclusion, ML365 provides a structural reference for future design of selective TWIK2 channel inhibitors in treating related inflammatory diseases.

CDER167, a dual inhibitor of URAT1 and GLUT9, is a novel and potent uricosuric candidate for the treatment of hyperuricemia.

Urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) are important targets for the development of uric acid-lowering drugs. We previously showed that the flexible linkers of URAT1 inhibitors could enhance their potency. In this study we designed and synthesized CDER167, a novel RDEA3710 analogue, by introducing a linker (methylene) between the naphthalene and pyridine rings to increase flexibility, and characterized its pharmacological and pharmacokinetics properties in vitro and in vivo. We showed that CDER167 exerted dual-target inhibitory effects on both URAT1 and GLUT9: CDER167 concentration-dependently inhibited the uptake of [14C]-uric acid in URAT1-expressing HEK293 cells with an IC50 value of 2.08 ± 0.31 µM, which was similar to that of RDEA3170 (its IC50 value was 1.47 ± 0.23 µM). Using site-directed mutagenesis, we demonstrated that CDER167 might interact with URAT1 at S35 and F365. In GLUT9-expressing HEK293T cells, CDER167 concentration-dependently inhibited GLUT9 with an IC50 value of 91.55 ± 15.28 µM, whereas RDEA3170 at 100 µM had no effect on GLUT9. In potassium oxonate-induced hyperuricemic mice, oral administration of CDER167 (10 mg·kg-1 · d-1) for 7 days was more effective in lowering uric acid in blood and significantly promoted uric acid excretion in urine as compared with RDEA3170 (20 mg·kg-1 · d-1) administered. The animal experiment proved the safety of CDER167. In addition, CDER167 displayed better bioavailability than RDEA3170, better metabolic stability and no hERG toxicity at 100 µM. These results suggest that CDER167 deserves further investigation as a candidate antihyperuricemic drug targeting URAT1 and GLUT9.

Apigenin ameliorates hyperuricemic nephropathy by inhibiting URAT1 and GLUT9 and relieving renal fibrosis via the Wnt/ß-catenin pathway.

BACKGROUND: Hyperuricemia (HUA) is characterized by abnormal serum uric acid (UA) levels and demonstrated to be involved in renal injury leading to hyperuricemic nephropathy (HN). Apigenin (API), a flavonoid naturally present in tea, berries, fruits, and vegetables, exhibits various biological functions, such as antioxidant and anti-inflammatory activity. PURPOSE: To investigate the effect of API treatment in HN and to reveal its underlying mechanisms. METHODS: The mice with HN were induced by potassium oxonate intraperitoneally and orally administered for two weeks. The effects of API on renal function, inflammation, fibrosis, and uric acid (UA) metabolism in mice with HN were evaluated. The effects of API on urate transporters were further examined in vitro. RESULTS: The mice with HN exhibited abnormal renal urate excretion and renal dysfunction accompanied by increased renal inflammation and fibrosis. In contrast, API reduced the levels of serum UA, serum creatinine (CRE), blood urea nitrogen (BUN) and renal inflammatory factors in mice with HN. Besides, API ameliorated the renal fibrosis via Wnt/ß-catenin pathway suppression. Furthermore, API potently promoted urinary UA excretion and inhibited renal urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) in mice with HN. In vitro, API competitively inhibited URAT1 and GLUT9 in a dose-dependent manner, with IC50 values of 0.64 ± 0.14 µM and 2.63 ± 0.69 µM, respectively. CONCLUSIONS: API could effectively attenuate HN through co-inhibiting UA reabsorption and Wnt/ß-catenin pathway, and thus it might be a potential therapy to HN.

Characterizations of the Urate Transporter, GLUT9, and Its Potent Inhibitors by Patch-Clamp Technique.

Glucose transporter 9 (GLUT9), which transports urate in an electrogenic and voltage-dependent manner, plays an important role in the maintenance of normal blood uric acid/urate levels. In the present study, we established a cell model based on the single-electrode patch-clamp technique for characterization of GLUT9 and explored the inhibitory effects of benzobromarone (BM) and probenecid (PB) on urate-induced currents in mouse GLUT9a (mGLUT9a)-expressing HEK-293T cells. The results showed that uric acid, rather than glucose perfusion, led to a rapid and large outward current by mGLUT9a in dose-, voltage-, and pH-dependent manners. BM prominently and irreversibly inhibited the uric acid-induced currents through mGLUT9a, and PB weakly and reversibly inhibited mGLUT9a. We found that depletion of K+ in the external solution significantly strengthened the blockade of BM on mGLUT9a. In addition, an enhanced inhibitory rate of BM was detected when the pH of the external solution was changed from 7.4 to 5.5, indicating that BM functions optimally in an acidic environment. In conclusion, the combination of the established cell model with patch-clamp techniques first revealed the function properties of GLUT9 inhibitors and may provide potential benefits to the study of GLUT9 inhibitors as antihyperuricemic or antigout agents.

Baicalein alleviates hyperuricemia by promoting uric acid excretion and inhibiting xanthine oxidase.

BACKGROUND: Insufficient renal urate excretion and/or overproduction of uric acid (UA) are the dominant causes of hyperuricemia. Baicalein (BAL) is widely distributed in dietary plants and has extensive biological activities, including antioxidative, anti-inflammatory and antihypertensive activities. PURPOSE: To investigate the anti-hyperuricemic effects of BAL and the underlying mechanisms in vitro and in vivo. METHODS: We investigated the inhibitory effects of BAL on GLUT9 and URAT1 in vitro through electrophysiological experiments and 14C-urate uptake assays. To evaluate the impact of BAL on serum and urine UA, the expression of GLUT9 and URAT1, and the activity of xanthine oxidase (XOD), we developed a mouse hyperuricemia model by potassium oxonate (PO) injection. Molecular docking analysis based on homology modeling was performed to explain the predominant efficacy of BAL compared with the other test compounds. RESULTS: BAL dose-dependently inhibited GLUT9 and URAT1 in a noncompetitive manner with IC50 values of 30.17 ± 8.68 µM and 31.56 ± 1.37 µM, respectively. BAL (200 mg/kg) significantly decreased serum UA and enhanced renal urate excretion in PO-induced hyperuricemic mice. Moreover, the expression of GLUT9 and URAT1 in the kidney was downregulated, and XOD activity in the serum and liver was suppressed. The docking analysis revealed that BAL potently interacted with Trp336, Asp462, Tyr71 and Gln328 of GLUT9 and Ser35 and Phe241 of URAT1. CONCLUSION: These results indicated that BAL exerts potent antihyperuricemic efects through renal UA excretal promotion and serum UA production. Thus, we propose that BAL may be a promising treatment for the prevention of hyperuricemia owing to its multitargeted inhibitory activity.

T-type calcium channels blockers inhibit HSV-2 infection at the late stage of genome replication.

Herpes simplex virus type 2 (HSV-2) is a highly contagious sexually transmitted virus. The increasing emergence of drug-resistant viral strains has highlighted the crucial need for the development of new anti-HSV-2 drugs with different mechanisms. Ion channels that govern a wide range of cellular functions represent attractive targets for viral manipulation. Here, we tried to identify novel compounds to suppress HSV-2 infection in vitro by screening a small library with ion channels modulators. We found that several T-type calcium channel blockers including benidipine, lercanidipine, lomerizine and mibefradil inhibited HSV-2 infection, while L-type calcium channel blockers nifedipine and nitrendipine showed no significant effect on HSV-2 infection. Furthermore, we found that benidipine exerted the antiviral effect by suppressing the expression of viral genes in the late stage of viral infection. In conclusion, our study suggested that T-type calcium channel blockers, which are clinically wide used, could effectively inhibit HSV-2 infection. These findings could shed light on the mechanism and pharmacological study for HSV-2 infection in the future.

Inhibition of hyperpolarization-activated cyclic nucleotide-gated channels with natural flavonoid quercetin.

Quercetin is a natural flavonoid which has been reported to be analgesic in different animal models of pain. However, the mechanism underlying the pain-relieving effects is still unclear. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in controlling pacemaker activity in cardiac and nervous systems, making the channel a new target for therapeutic exploration. In this study, we explored a series of flavonoids for their modulation on HCN channels. Among all tested flavonoids, quercetin was the most potent inhibitor for HCN channels with an IC50 value of 27.32 ± 1.19 µM for HCN2. Furthermore, quercetin prominently left shifted the voltage-dependent activation curves of HCN channels and decelerated deactivation process. The results presented herein firstly characterize quercetin as a novel and potent inhibitor for HCN channels, which represents a novel structure for future drug design of HCN channel inhibitors.

Distinct Molecular Mechanisms Underlying Potassium Efflux for NLRP3 Inflammasome Activation.

The NLRP3 inflammasome is a core component of innate immunity, and dysregulation of NLRP3 inflammasome involves developing autoimmune, metabolic, and neurodegenerative diseases. Potassium efflux has been reported to be essential for NLRP3 inflammasome activation by structurally diverse pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Thus, the molecular mechanisms underlying potassium efflux to activate NLRP3 inflammasome are under extensive investigation. Here, we review current knowledge about the distinction channels or pore-forming proteins underlying potassium efflux for NLRP3 inflammasome activation with canonical/non-canonical signaling or following caspase-8 induced pyroptosis. Ion channels and pore-forming proteins, including P2X7 receptor, Gasdermin D, pannexin-1, and K2P channels involved present viable therapeutic targets for NLRP3 inflammasome related diseases.

Structural Insights into the Atomistic Mechanisms of Uric Acid Recognition and Translocation of Human Urate Anion Transporter 1.

Background: Human urate transporter 1 (hURAT1) is the most pivotal therapeutic target for treating hyperuricemia. However, the molecular interactions between uric acid and URAT1 are still unknown due to lack of structural details. Methods: In the present study, several methods (homology modeling, sequence alignment, docking, and mutagenesis) were used to explain the atomistic mechanisms of uric acid transport of hURAT1. Results: Residues W357-F365 in the TMD7 and P484-R487 in the TMD11 present in the hURAT1 have unique roles in both binding to the uric acid and causing subsequent structural changes. These residues, located in the transport tunnel, were found to be related to the structural changes, as demonstrated by the reduced V max values and an unaltered expression of protein level. In addition, W357, G361, T363, F365, and R487 residues may confer high affinity for binding to uric acid. An outward-open homology model of hURAT1 revealed a crucial role for these two domains in the conformational changes of hURAT1. F241 and H245 in TMD5, and R477 and R487 in TMD11 may confer high affinity for uric acid, and as the docking analysis suggests, they may also enhance the affinity for the inhibitors. R477 relation to the structural changes was demonstrated by the V max values of the mutants and the contribution of positive charge to the uric acid selectivity. Conclusions: W357-F365 in TMD7, P484-R487 in TMD11, and residues F241, H245, and R477 were found to be critical for the translocation and recognition of uric acid.

Resveratrol promotes HSV-2 replication by increasing histone acetylation and activating NF-κB.

Resveratrol is a natural compound found in many plant species that has broad therapeutic benefits. Here, we investigated the effects of resveratrol on the replication of HSV-2. We found that resveratrol accelerated replication of HSV-2 and increased release of progeny virion. A time-of-addition study suggested that resveratrol worked primarily in the early stage of viral infection. Resveratrol regulated HSV-2 infection by increasing histone acetylation and activating NF-κB. In addition, inhibition of CDK9 activity restrained the promoting effect of resveratrol on HSV-2 infection. Altogether, our experiments revealed the regulatory effect of resveratrol and its mechanism of action on HSV-2 replication.