Therapeutic effects on H1N1-induced pneumonia in mice and intestinal bacteria biotransformation of four main flavonoids from Houttuynia cordata Thunb.

Flavonoids widely exist in a large number of Chinese herbal medicines with antiviral and anti-inflammatory properties. Houttuynia cordata Thunb. is a traditional Chinese herbal medicine for heat-clearing and detoxification. In our previous research, total flavonoids from H. cordata (HCTF) effectively alleviated H1N1-induced acute lung injury (ALI) in mice. In this study, 8 flavonoids were recognized from HCTF (containing 63.06 % ± 0.26 % of total flavonoids, as quercitrin equivalents) by UPLC-LTQ-MS/MS. Four main flavonoid glycosides in HCTF (rutin, hyperoside, isoquercitrin and quercitrin) and their common aglycone quercetin (100 mg/kg) all showed therapeutic effects on H1N1-induced ALI in mice. The two flavonoids (hyperoside and quercitrin) with higher contents and quercetin showed stronger therapeutic effects on H1N1-induced ALI in mice. Hyperoside, quercitrin and quercetin significantly reduced the levels of pro-inflammatory factors, chemokines, or neuraminidase activity compared with the same dose of HCTF (p < 0.05). The results of mice intestinal bacteria biotransformation in vitro showed that quercetin was the main metabolite. The conversion rates of hyperoside and quercitrin were significantly higher by the intestinal bacteria under the pathological state (0.81 ± 0.02 and 0.91 ± 0.01, respectively) than normal state (0.18 ± 0.01 and 0.18 ± 0.12, respectively, p < 0.001). Our findings showed that hyperoside and quercitrin were the main efficacious components of HCTF for treating H1N1-induced ALI in mice and could be metabolized to quercetin by intestinal bacteria in pathological state to exert their effects.

Vunakizumab-IL22, a Novel Fusion Protein, Promotes Intestinal Epithelial Repair and Protects against Gut Injury Induced by the Influenza Virus.

Secondary immune damage to the intestinal mucosa due to an influenza virus infection has gained the attention of investigators. The protection of the intestinal barrier is an effective means of improving the survival rate in cases of severe pneumonia. We developed a fusion protein, Vunakizumab-IL22(vmab-IL22), by combining an anti-IL17A antibody with IL22. Our previous study showed that Vunakizumab-IL22 repairs the pulmonary epithelial barrier in influenza virus-infected mice. In this study, we investigated the protective effects against enteritis given its anti-inflammatory and tissue repair functions. The number of goblet cells and the expression of zonula occludens protein 1(ZO-1), Mucin-2, Ki67 and IL-22R were determined by immunohistochemistry (IHC) and quantitative RT-PCR in influenza A virus (H1N1)-infected mice. The expression of NOD-like receptor pyrin domain containing 3 (NLRP3) and toll- like-receptor-4 (TLR4) was assayed by IHC in the lungs and intestine in HIN1 virus-induced mice to evaluate the whole efficacy of the protective effects on lungs and intestines. Consequently, Cytochrome C, phosphorylation of nuclear factor NF-kappaB (p-NF-κB), IL-1ß, NLRP3 and Caspase 3 were assayed by Western blotting in dextran sulfate sodium salt (DSS)-treated mice. Treatment with Vunakizumab-IL22 improved the shortened colon length, macroscopic and microscopic morphology of the small intestine (p < 0.001) significantly, and strengthened the tight junction proteins, which was accompanied with the upregulated expression of IL22R. Meanwhile, Vunakizumab-mIL22 inhibited the expression of inflammation-related protein in a mouse model of enteritis induced by H1N1 and DSS. These findings provide new evidence for the treatment strategy for severe viral pneumonia involved in gut barrier protection. The results suggest that Vunakizumab-IL22 is a promising biopharmaceutical drug and is a candidate for the treatment of direct and indirect intestinal injuries, including those induced by the influenza virus and DSS.

The protective effect of 999 XiaoErGanMao granules on the lungs and intestines of influenza A virus-infected mice.

CONTEXT: Gastrointestinal symptoms are a common complication of influenza virus infection in children, which the gut-lung axis become involved in its biological progress. The protective effect of 999 XiaoErGanMao granules (XEGMG) on multi-organ injury in viral pneumonia remains unclear. OBJECTIVE: To investigate the therapeutic effect of XEGMG on lungs and intestines injury in A/FM/1/47 (H1N1) influenza virus-infected mice. MATERIALS AND METHODS: Male BALB/c mice were infected with the 2LD50 H1N1 influenza virus and then treated with XEGMG (6 or 12 g/kg) intragastrically once a day for 4 days. The lung and colon samples were then collected for pathological observation, and assays for inflammatory cytokines and intestinal barrier. Mouse feces were collected to evaluate the intestinal microbiota. RESULTS: Treating with XEGMG (12 g/kg) can mitigate body weight loss caused by 2LD50 H1N1 infection. It can also reduce lung index and pathological damage with the decreased inflammatory cytokines such as IL-6 and IL-1ß. Furthermore, XEGMG (12 g/kg) can maintain the goblet cell number in the colons to protect the intestinal barrier and regulate the major flora such as Firmicutes, Bacteroidetes, and Muribaculaceae back to normal. Meanwhile, the expression of IL-17A in the colon tissues was significantly lower in the group of XEGMG (6, 12 g/kg) compared to H1N1 group. DISCUSSION AND CONCLUSIONS: XEGMG can protect against H1N1 invasion involved in gut-lung axis regulation. The results provide new evidence for the protective effect of XEGMG, which is beneficial to vulnerable children.

Targeting CD47 enhanced the antitumor immunity of PD-L1 blockade in B-cell lymphoma.

Background: Only a subset of B-cell lymphoma (BCL) patients can benefit from immune checkpoint inhibitors targeting PD-1/PD-L1. Materials & methods: In the A20 model, SIRPα-Fc and anti-PD-L1 were employed to target CD47 and PD-L1 simultaneously. Flow cytometry, immunofluorescence and quantitative polymerase chain reaction were used to unravel the potential mechanisms. Results: Simultaneously targeting CD47 and PD-L1 activated CD8+ T cells with an increased release of effector molecules. Furthermore, infiltration of F4/80+iNOS+ M1 macrophages was enhanced by the dual therapy. Conclusion: Anti-CD47 therapy could sensitize BCL tumors to anti-PD-L1 therapy in a CD8+ T-cell- and M1-macrophage-dependent manner by promoting cytotoxic lymphocyte infiltration, which may provide a potential strategy for BCL treatment by simultaneously targeting CD47 and PD-L1.

Immune checkpoint inhibitors targeting PD-1/PD-L1 have become effective agents for cancer treatment. However, only a minority of patients benefit from this treatment in the clinic because of the limited response rate. Targeting CD47/SIRPα restores macrophage function and improves the response of antitumor immunity. Here, combination immunotherapy targeting CD47/SIRPα and PD-1/PD-L1 was investigated to increase the response rate and antitumor effect of PD-L1 monotherapy in B-cell lymphoma (BCL). This study broadens the application of the combination therapy and provided a promising strategy for B-cell lymphoma treatment by simultaneous targeting of PD-1/PD-L1 and CD47/SIRPα axis.

Benvitimod inhibits MCM6-meditated proliferation of keratinocytes by regulating the JAK/STAT3 pathway.

BACKGROUND: Benvitimod (Tapinarof), as a small-molecule topical therapeutical aryl hydrocarbon receptor (AHR)-modulating agent, is in clinical development for treating psoriasis and atopic dermatitis. Benvitimod reduces proinflammatory cytokines in psoriasis by specifically binding and activation of AHR. However, whether benvitimod can inhibit keratinocyte proliferation remains unclear. Minichromosome maintenance protein 6 (MCM6) is a key element of the prereplication complex (pre-RC) assembly which is one of the essential steps in the initiation of DNA replication for cell proliferation. OBJECTIVES: This study aimed to determine whether benvitimod could reduce the excessive proliferation of psoriatic keratinocytes by inhibiting MCM6. METHODS: We examined the inhibitory effect of benvitimod on MCM6-mediated proliferation of keratinocytes by HaCaT cells in vitro and an IMQ-induced psoriatic model of mice in vivo. RESULTS: Epidermal MCM6 expression was enhanced in the skin lesions of psoriatic patients. The experiments further revealed that MCM6 was required for the proliferation of keratinocytes and governed by the IL-22/STAT3 pathway. In addition, the antiproliferation effect of benvitimod is achieved by the inhibition of p-JAK1 and p-JAK2, which further restrained the activation of STAT3 in keratinocytes. Lastly, benvitimod could repressed imiquimod-induced skin lesions and the expression of epidermal MCM6 and p-STAT3 in mice. Moreover, knockdown of AHR in keratinocytes enhanced the activation of JAK1 and JAK2. CONCLUSION: The findings reveal that benvitimod could decrease MCM6-mediated proliferation of keratinocytes by affecting the JAK/STAT3 pathway, thereby serving as a new treatment modality for psoriasis.

Houttuynia cordata polysaccharides alleviate ulcerative colitis by restoring intestinal homeostasis.

Houttuynia cordata is traditionally used as phytoantibiotics for treating lung disease in China. Houttuynia cordata polysaccharides (HCPs) have been reported to alleviate influenza virus-induced intestinal and lung immune injury by regulating the gut-lung axis. The present study aims to investigate the effects and mechanisms of HCPs on ulcerative colitis (UC). Male C57BL/6 mice were induced by dextran sodium sulfate (DSS) to establish the UC animal model. Our results showed that HCPs significantly reduced the weight loss and the shortening of colon length in colitis mice, and relieved the pathological damage of colon mucosa and inhibited the expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6, etc. It was suggested that HCPs could significantly improve DSS-induced colitis in mice. HCPs directly protected intestinal epithelial cells, ameliorated epithelial barrier dysfunction and cell apoptosis, which was also proved in H2O2 stimulated cell apoptosis model. HCPs inhibited inflammation in the colon, which was related to suppressing the infiltration of macrophages, inhibiting the expression of pro-inflammatory cytokines and proteins (TLR4, NF-κB), and restoring the dysfunction of Th17 and Treg cells. HCPs also restored the alteration of intestinal flora induced by DSS, increased the abundance ofFirmicutes and Bacteroides, and reduced the abundance of Proteobacteria. This study confirmed the protective effect of Houttuynia cordata polysaccharide extracted from traditional Chinese medicine on ulcerative colitis, of which the mechanism was closely related to the maintenance of intestinal homeostasis (intestinal barrier, immune cells, and intestinal bacteria).

Therapeutic Potential of 2-Methylquinazolin-4(3H)-one as an Antiviral Agent against Influenza A Virus-Induced Acute Lung Injury in Mice.

Qingdai-Mabo (QM), a traditional Chinese herbal formula composed of medicinal herb and fungus, has been used for treatment of cough and viral pneumonia. However, the underlying mechanism and bioactive components against anti-influenza A virus remain unclear. In the present study, ethyl acetate (EA) extract of QM decoctions was tested for its biological activity against acute lung injury (ALI) and its main components were identified using UPLC-MS/MS. In total, 18 bioactive components were identified, including 2-Methylquinaozlin-4(3H)-one (C1), which showed significant antiviral activity in vitro with an IC50 of 23.8 µg/mL. Furthermore, we validated the efficacy of C1 in ameliorating ALI lesions and inflammation in influenza A virus-infected mice. The results showed that C1 significantly reduced the lung index, downregulated neuraminidase (NA) and nucleoprotein (NP), and decreased the expression of pro-inflammatory molecules IFN-α, TNF-α, MCP-1, IL-6, and IL-8; however, they enhanced levels of IL-10 and IFN-γ in lung homogenate from mice infected by influenza A virus. In addition, C1 inhibited the recruitment of macrophages. These in vitro and in vivo studies suggested that the significant anti-influenza A virus activity contributed to its curative effect on lesions and inflammation of viral pneumonia in mice. Given its potential antiviral activity against influenza A virus, C1 is determined to be a main active component in the EA extract of QM. Taken together, the antiviral activity of C1 suggests its potential as an effective treatment against viral pneumonia via the inhibition of virus replication, but the mechanism C1 on antiviral research needs to be explored further.

Intestinal microbiota metabolizing Houttuynia cordata polysaccharides in H1N1 induced pneumonia mice contributed to Th17/Treg rebalance in gut-lung axis.

Influenza A virus is intricately linked to dysregulation of gut microbiota and host immunity. Previous study revealed that Houttuynia cordata polysaccharides (HCP) exert the therapeutic effect on influenza A virus inducing lung and intestine damage via regulating pulmonary and intestinal mucosal immunity. However, whether this result was due to the regulation of gut microbiota in the gut-lung axis remains unclear. Here, we firstly found that the elimination of gut microbiota using antibiotic cocktails led to both loss of the protective effect of HCP on intestine and lung injury, and reduction of the efficacy on regulating Th17/Treg balance in gut-lung axis. Fecal microbiota transplantation study confirmed that the gut microbiota fermented with HCP under pathological conditions (H1N1 infection) was responsible for reducing pulmonary and intestinal injury. Moreover, the interaction of HCP and gut microbiota under pathological conditions exhibited not only much more abundant gut microbial diversity, but also higher content of the acetate. Our results demonstrated that the underlying mechanism to ameliorate viral pneumonia in mice involving Th17/Treg rebalance via the gut microbiota and HCP metabolite (acetate) metabolized in pneumonia mice. Our results provided a new insight for macromolecular polysaccharides through targeting intestinal microenvironment reducing distant pulmonary infection.

Integrated network pharmacology and intestinal flora analysis to determine the protective effect of Xuanbai-Chengqi decoction on lung and gut injuries in influenza virus-infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanbai-Chengqi decoction (XBCQ) is a traditional Chinese medicine (TCM) compound used in the treatment of pulmonary infection in China. Despite the popular usage of XBCQ, its underlying protective roles and the associated molecular mechanisms with the gut-lung axis in influenza remain unclear. AIM OF THE STUDY: We aimed to explore the protective effects and the underlying mechanism of XBCQ efficacy on lung and intestine injuries induced by influenza A virus as well as to identify the main active components through integrated network pharmacology, intestinal flora analysis and pathway validation. MATERIALS AND METHODS: The potential active components and therapeutic targets of XBCQ in the treatment of influenza were hypothesized through a series of network pharmacological strategies, including components screening, targets prediction and bioinformatics analysis. Inflammatory cytokines and pathway proteins were assayed to validate the results of network pharmacology. Then the mechanism of XBCQ alleviating lung and intestine injuries was further explored via intestinal flora analysis. The important role of Rhubarb in the formula was verified by removing Rhubarb. RESULTS: XBCQ could significantly improve the survival rate in IAV-infected mice. The network pharmacology results demonstrated that JUN, mitogen-activated protein kinase (MAPK), and tumor necrosis factor (TNF) are the key targets of XBCQ that can be useful in influenza treatment as it contains the core components luteolin, emodin, and aloe-emodin, which are related to the pathways of TNF, T-cell receptor (TCR), and NF-κB. Verification experiments demonstrated that XBCQ could significantly alleviate the immune injury of the lungs and the gut of the mice, which is attributable to the inhibition of the release of inflammatory cytokines (such as TNF-α, IL-6, and IL-1ß), the downregulation of the protein expression levels of Toll-like receptors-7 (TLR7), MyD88, and p-NF-κB65, and the reduction in the relative abundance of Enterobacteriaceae and Proteus, while an increase in that of Firmicutes and Lachnospiraceae. The overall protective role of XBCQ contributing to the treatment of the lungs and the gut was impaired when Rhubarb was removed from XBCQ. CONCLUSIONS: Our results suggest that the efficacy of XBCQ is related to the inhibition of the immune injury and remodeling of the intestinal flora, wherein Rhubarb plays an important role, which cumulatively provide the evidence applicable for the treatment of viral pneumonia induced by a different respiratory virus with XBCQ.

Neutrophil extracellular traps mediate severe lung injury induced by influenza A virus H1N1 in mice coinfected with Staphylococcus aureus.

Influenza virus and bacterial infection contributed to massive morbidity and mortality. However, the underlying mechanisms were poorly understood. A coinfected model was generating by using sublethal doses of influenza A virus H1N1 A/FM/1/47(H1N1) and methicillin-resistant Staphylococcus aureus (MRSA). Further, the model was optimized to achieve the highest peak of mortality initiated by intranasal infection with 0.2LD50 H1N1 and 0.16LD50 MRSA at 3 days interval. Excessive neutrophil recruitment, accompanied by high levels of inflammatory cytokines and chemokines, and increased bacterial and viral load were observed in coinfected mice. Under the inflammatory environments triggered by H1N1 and MRSA, the excessive neutrophil recruitment led to the formation of neutrophil extracellular traps (NETs), associated with severe inflammation and vascular endothelial injury. Importantly, the severity of lung injury could be alleviated by treatment with DNase I or a selective neutrophil elastase inhibitor (NEi). Therefore, our data suggested that excessive neutrophil recruitment and NETs formation contributed to severe inflammation and acute lung injury in coinfected animals.

Liver-Targeted Nanoparticles Facilitate the Bioavailability and Anti-HBV Efficacy of Baicalin In Vitro and In Vivo.

The anti-hepatitis B virus (HBV) efficacy of baicalin (BA) is mediated by HBV-related hepatocyte nuclear factors (HNFs). However, this efficacy is severely limited by the low bioavailability of BA. Therefore, a novel liver-targeted BA liposome was constructed to promote the bioavailability and antiviral ability of BA. The results showed that apolipoprotein A1 (ApoA1)-modified liposomes (BAA1) significantly enhanced BA's cellular uptake and specific distribution in the liver. Furthermore, the substantial inhibitory effects of BAA1 on HBsAg, HBeAg, HBV RNA, and HBV DNA were assessed in HB-infected cells and mice. Western blotting, co-immunoprecipitation, and transcriptomics analysis further revealed that the enhanced anti-HBV efficacy of BAA1 was attributed to the interaction between hepatocyte nuclear factors (HNFs) and estrogen receptors (ERs). Based on the findings, we propose that the ApoA1-modified liposomes aid BA in inhibiting HBV transcription and replication by augmenting its bioavailability and the HNFs-ERs axis.

Autophagy and Exosome Coordinately Enhance Macrophage M1 Polarization and Recruitment in Influenza A Virus Infection.

Background: Influenza A virus infection results in viral pneumonia, which is often accompanied by the infiltration and recruitment of macrophages, overactivation of inflammatory responses, and obvious cell autophagy and exosome production. However, little is known about the roles of autophagy and exosome production in these inflammatory responses. Methods: In this study, multiple methods, such as flow cytometry, real-time quantitative reverse transcription-polymerase chain reaction, immune-fluorescence technology, and western blot, were applied to explore the possible effects of autophagy and exosome production by H1N1-infected host cells. Results: It was observed that a high number of polarized macrophages (CD11b+/F4/80+/CD86+) were recruited to the lung tissues of infected mice, which could be mimicked by tracking the movement of macrophages to H1N1-infected cells in vitro (transwell assays). Furthermore, there was some coordinated upregulation of M1 polarization signs (iNOS/Arg-1 bias) as well as autophagy (LC3) and exosome (CD63) biomarkers in the infected macrophages and epithelial cells. Moreover, exosomes extracted from the supernatant of virus-infected cells were shown to promote the recruitment and polarization of more peritoneal macrophages than the normal group. The fluorescence colocalization of LC3-CD63 and the inhibition of autophagy and exosome signaling pathway further revealed that H1N1 infection seemed to sequentially activate the M1 polarization and recruitment of macrophages via autophagy-exosome dependent pathway. Conclusion: Autophagy and exosome production coordinately enhance the M1 polarization and recruitment of macrophages in influenza virus infection, which also provides potential therapeutic targets.

Systematic optimization of host-directed therapeutic targets and preclinical validation of repositioned antiviral drugs.

Inhibition of host protein functions using established drugs produces a promising antiviral effect with excellent safety profiles, decreased incidence of resistant variants and favorable balance of costs and risks. Genomic methods have produced a large number of robust host factors, providing candidates for identification of antiviral drug targets. However, there is a lack of global perspectives and systematic prioritization of known virus-targeted host proteins (VTHPs) and drug targets. There is also a need for host-directed repositioned antivirals. Here, we integrated 6140 VTHPs and grouped viral infection modes from a new perspective of enriched pathways of VTHPs. Clarifying the superiority of nonessential membrane and hub VTHPs as potential ideal targets for repositioned antivirals, we proposed 543 candidate VTHPs. We then presented a large-scale drug-virus network (DVN) based on matching these VTHPs and drug targets. We predicted possible indications for 703 approved drugs against 35 viruses and explored their potential as broad-spectrum antivirals. In vitro and in vivo tests validated the efficacy of bosutinib, maraviroc and dextromethorphan against human herpesvirus 1 (HHV-1), hepatitis B virus (HBV) and influenza A virus (IAV). Their drug synergy with clinically used antivirals was evaluated and confirmed. The results proved that low-dose dextromethorphan is better than high-dose in both single and combined treatments. This study provides a comprehensive landscape and optimization strategy for druggable VTHPs, constructing an innovative and potent pipeline to discover novel antiviral host proteins and repositioned drugs, which may facilitate their delivery to clinical application in translational medicine to combat fatal and spreading viral infections.

Dimeric clerodane diterpenoids and antiviral constituents of Dodonaea viscosa.

Three unprecedented dimeric clerodane diterpenoids, dodovisdimers A-C (1-3), along with six known clerodane monomers (4-9), were isolated from Dodonaea viscosa. Compounds 1-3 may be biosynthetically formed via an intermolecular Diels-Alder [4+2] cycloaddition between the coexisting monomers 4-7. The structures of these clerodanes were characterized by spectroscopic techniques, X-ray crystallographic study, and ECD calculations. Some isolates exerted antiviral effects on human influenza A virus (H3N2) in vitro.

Polymorphic Characterization, Pharmacokinetics, and Anti-Inflammatory Activity of Ginsenoside Compound K Polymorphs.

Polymorphism exhibits different physicochemical properties, which can impact the bioavailability and bioactivity of solid drugs. This study focused on identifying the polymorphs of ginsenoside compound K (CK) and studying their different behaviors in pharmacokinetics (PK) and pharmacodynamics (PD). Four CK polymorphs (form I, II, III, and IV) from organic solvents were characterized by scanning electron microscope (SEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and powder X-ray diffraction (PXRD). A feasible LC-MS/MS method was exploited to determine the PK parameters. Form II displayed the most exposure, followed by form I, III, and IV. Notably, all forms showed sex dimorphism, and the bioavailability in the female group was about two-fold higher than in the male group. The PD properties were investigated in carrageenan-induced acute paw inflammation, and form II at 20 mg/kg showed significant inhibition of edema by 42.7%. This study clarified the polymorphic, PK, and PD characters of four crystal forms of CK, and the data suggested that form II had the best efficacy for drug development.

Baicalin Inhibits Influenza A Virus Infection via Promotion of M1 Macrophage Polarization.

BACKGROUND AND AIMS: The natural compound baicalin (BA) possesses potent antiviral properties against the influenza virus. However, the underlying molecular mechanisms of this antiviral activity and whether macrophages are involved remain unclear. In this study, we, therefore, investigated the effect of BA on macrophages. METHODS: We studied macrophage recruitment, functional phenotypes (M1/M2), and the cellular metabolism via flow cytometry, qRT-PCR, immunofluorescence, a cell culture transwell system, and GC-MS-based metabolomics both in vivo in H1N1 A virus-infected mice and in vitro. RESULTS: BA treatment drastically reduced macrophage recruitment (CD11b+, F4/80+) by approximately 90% while maintaining the proportion of M1-polarized macrophages in the bronchoalveolar lavage fluid of infected mice. This BA-stimulated macrophage M1 phenotype shift was further verified in vitro in ANA-1 and primary peritoneal macrophages by measuring macrophage M1 polarization signals (CD86, iNOS, TNF-α, iNOS/Arg-1 ratio, and IL-1ß cleavage). Meanwhile, we observed an activation of the IFN pathway (upregulation of IFN-ß and IRF-3), an inhibition of influenza virus replication (as measured by the M gene), and distinct cellular metabolic responses in BA-treated cells. CONCLUSION: BA triggered macrophage M1 polarization, IFN activation, and other cellular reactions, which are beneficial for inhibition of H1N1 A virus infection.

Baicalin down-regulating hepatitis B virus transcription depends on the liver-specific HNF4α-HNF1α axis.

Baicalin (BA) inhibits hepatitis B virus (HBV) RNAs production and reduces levels of the related hepatocyte nuclear factors (HNFs), although the underlying mechanism is unclear. In this study, we investigated the specific pathway by which BA regulates HBV transcription through the HBV-related HNFs. Following transfection of HepG2 cells with pHBV1.2, we observed that BA inhibited the production of HBV RNAs and viral proteins in a time- and dose-dependent manner. These effects were consistent with the downregulation of HNF1α, which was abolished by HNF1α-shRNA. The shRNA of HNF4α, the upstream gene of HNF1α, also remarkedly reduced HNF1α expression and impaired the anti-HBV efficacy of BA, indicating that this function of BA depended on HNF4α/HNF1α axis. Furthermore, chromatin immunoprecipitation assay showed that BA significantly reduced HNF4α-HNF1α transactivation activity. The similar effects of BA were observed in entecavir (ETV)-resistant HBVrtM204V/rtLl80M transfected HepG2 cells. Thus, we proposed a mechanism for the anti-HBV activity of BA in an HNF4α-HNF1α-dependent manner, which impaired HNF4α and HNF1α transactivation, and effectively inhibited HBV transcription and viral replication.

Regulating the balance of Th17/Treg cells in gut-lung axis contributed to the therapeutic effect of Houttuynia cordata polysaccharides on H1N1-induced acute lung injury.

Our previous study had demonstrated that oral administration of Houttuynia cordata polysaccharides (HCP) without in vitro antiviral activity ameliorated gut and lung injuries induced by influenza A virus (IAV) in mice. However, as macromolecules, HCP was hard to be absorbed in gastrointestinal tract and had no effect on lung injury when administrated intravenously. The action mechanism of HCP was thus proposed as regulating the gut mucosal-associated lymphoid tissue (GALT). Actually, HCP treatment restored the balance of Th17/Treg cells firstly in GALT and finally in the lung. HCP reduced the expression of chemokine CCL20 in the lung and regulated the balance of Th17/Treg carrying CCR6+ (the CCL20 receptor), which was associated with specific migration of Th17/Treg cells from GALT to lung. In vitro, HCP inhibited Th17 cell differentiation through the downregulation of phospho-STAT3, whereas it promoted Treg cell differentiation by upregulating phospho-STAT5. Furthermore, its therapeutic effect was abolished in RORγt-/- or Foxp3-/- mice. These findings indicated that oral administration of macromolecular polysaccharides like HCP might ameliorate lung injury in IAV infected mice via directly regulating the balance of Th17/Treg cells in gut-lung axis. Our results provided a potential mechanism underlying the therapeutic effect of polysaccharides on pulmonary infection.

Cycloartane triterpenoids from Pseudolarix amabilis and their antiviral activity.

Seven undescribed cycloartane triterpenoids, pseudolarnoids A-G, together with ten known ones, were isolated from the seeds of Pseudolarix amabilis (J. Nelson) Rehder. Their structures were elucidated on the basis of spectroscopic analysis, X-ray crystallography, and ECD data. Pseudolarnoids A-C are cycloartane triterpenoids with a unique 16S, 23R-spirolactone moiety. Pseudolarnoids F, G, and pseudolarolide C demonstrated potent antiviral effects on HSV-1 in vitro.

Chlorogenic acid protects PC12 cells against corticosterone-induced neurotoxicity related to inhibition of autophagy and apoptosis.

BACKGROUND: There are evidences that chlorogenic acid (CGA) has antidepressant effects, however the underlying molecular mechanism has not been well understood. The aim of the study was to explore the neuroprotective effect of CGA on corticosterone (CORT)-induced PC 12 cells and its mechanism, especially the autophagy pathway. METHODS: PC12 cells were incubated with CORT (0, 100, 200, 400 or 800 µM) for 24 h, cell viability was measured by MTT assay. PC12 cells were cultured with 400 µM of CORT in the absence or presence of CGA (25 µg/ml) for 24 h, morphologies and specific marker of autophagosome were observed by transmission electron microscope (TEM) and confocal immunofluorescence microscopy, respectively. In addition, PC12 cells were treated with different doses of CGA (0, 6.25, 12.5, 25 or 50 µg/ml) with or without CORT (400 µM) for 24 h, cell viability and changes in the morphology were observed, and further analysis of apoptotic and autophagic proteins, and expression of AKT/mTOR signaling pathway were carried out by Western blot. Specific inhibitors of autophagy 3-Methyladenine (3-MA) and chloroquine (CQ) were added to the PC12 cells cultures to explore the potential role of autophagy in CORT-induced neuronal cell apoptosis. RESULTS: Besides decreasing PC12 cell activity, CORT could also induce autophagy and apoptosis of PC12 cells, while CGA could reverse these effects. In addition, CGA treatment regulated AKT/mTOR signaling pathway in PC12 cells. CGA, similar to 3-MA and QC, significantly inhibited CORT-induced apoptosis in PC12 cells. CONCLUSIONS: Our results provide a new molecular mechanism for the treatment of CORT-induced neurotoxicity by CGA, and suggest CGA may be a potential substance which is can alleviate depression.