Platycodin D facilitates antiviral immunity through inhibiting cytokine storm via targeting K63-linked TRAF6 ubiquitination.

Influenza virus infection is a worldwide challenge that causes heavy burdens on public health. The mortality rate of severe influenza patients is often associated with hyperactive immunological abnormalities characterized by hypercytokinemia. Due to the continuous mutations and the occurrence of drug-resistant influenza virus strains, the development of host-directed immunoregulatory drugs is urgently required. Platycodon grandiflorum is among the top 10 herbs of traditional Chinese medicine used to treat pulmonary diseases. As one of the major terpenoid saponins extracted from Platycodon grandiflorum, Platycodin D (PD) has been reported to play several roles, including anti-inflammation, analgesia, anti-cancer, hepatoprotection, and immunoregulation. However, the therapeutic roles of PD to treat influenza virus infection remains unknown. Here, we show that PD can protect the body weight loss in severely infected influenza mice, alleviate lung damage, and thus improve the survival rate. More specifically, PD protects flu mice via decreasing the immune cell infiltration into lungs and downregulating the overactivated inflammatory response. Western blot and immunofluorescence assays exhibited that PD could inhibit the activation of TAK1/IKK/NF-κB and MAPK pathways. Besides that, CETSA, SPR and immunoprecipitation assays indicated that PD binds with TRAF6 to decrease its K63 ubiquitination after R837 stimulation. Additionally, siRNA interference experiments exhibited that PD could inhibit the secretion of IL-1ß and TNF-α in TRAF6-dependent manner. Altogether, our results suggested that PD is a promising drug candidate for treating influenza. Our study also offered a scientific explanation for the commonly used Platycodon grandiflorum in many anti-epidemic classic formulas. Due to its host-directed regulatory role, PD may serve as an adjuvant therapeutic drug in conjunction with other antiviral drugs to treat the flu.

Fei-Yan-Qing-Hua decoction decreases hyperinflammation by inhibiting HMGB1/RAGE signaling and promotes bacterial phagocytosis in the treatment of sepsis.

ETHNOPHARMACOLOGICAL RELEVANCE: Fei-Yan-Qing-Hua decoction (FYQHD), derived from the renowned formula Ma Xing Shi Gan tang documented in Zhang Zhong Jing's "Treatise on Exogenous Febrile Disease" during the Han Dynasty, has demonstrated notable efficacy in the clinical treatment of pneumonia resulting from bacterial infection. However, its molecular mechanisms underlying the therapeutic effects remains elusive. AIM OF THE STUDY: This study aimed to investigate the protective effects of FYQHD against lipopolysaccharide (LPS) and carbapenem-resistant Klebsiella pneumoniae (CRKP)-induced sepsis in mice and to elucidate its specific mechanism of action. MATERIALS AND METHODS: Sepsis models were established in mice through intraperitoneal injection of LPS or CRKP. FYQHD was administered via gavage at low and high doses. Serum cytokines, bacterial load, and pathological damage were assessed using enzyme-linked immunosorbent assay (ELISA), minimal inhibitory concentration (MIC) detection, and hematoxylin and eosin staining (H&E), respectively. In vitro, the immunoregulatory effects of FYQHD on macrophages were investigated through ELISA, MIC, quantitative real-time PCR (Q-PCR), immunofluorescence, Western blot, and a network pharmacological approach. RESULTS: The application of FYQHD in the treatment of LPS or CRKP-induced septic mouse models revealed significant outcomes. FYQHD increased the survival rate of mice exposed to a lethal dose of LPS to 33.3%, prevented hypothermia (with a rise of 3.58 °C), reduced pro-inflammatory variables (including TNF-α, IL-6, and MCP-1), and mitigated tissue damage in LPS or CRKP-induced septic mice. Additionally, FYQHD decreased bacterial load in CRKP-infected mice. In vitro, FYQHD suppressed the expression of inflammatory cytokines in macrophages activated by LPS or HK-CRKP. Mechanistically, FYQHD inhibited the PI3K/AKT/mTOR/4E-BP1 signaling pathway, thereby suppressing the translational level of inflammatory cytokines. Furthermore, it reduced the expression of HMGB1/RAGE, a positive feedback loop in the inflammatory response. Moreover, FYQHD was found to enhance the phagocytic activity of macrophages by upregulating the expression of phagocytic receptors such as CD169 and SR-A1. CONCLUSION: FYQHD provides protection against bacterial sepsis by concurrently inhibiting the inflammatory response and augmenting the phagocytic ability of immune cells.

Cytokine Storm in Acute Viral Respiratory Injury: Role of Qing-Fei-Pai-Du Decoction in Inhibiting the Infiltration of Neutrophils and Macrophages through TAK1/IKK/NF-[Formula: see text]B Pathway.

COVID-19 has posed unprecedented challenges to global public health since its outbreak. The Qing-Fei-Pai-Du decoction (QFPDD), a Chinese herbal formula, is widely used in China to treat COVID-19. It exerts an impressive therapeutic effect by inhibiting the progression from mild to critical disease in the clinic. However, the underlying mechanisms remain obscure. Both SARS-CoV-2 and influenza viruses elicit similar pathological processes. Their severe manifestations, such as acute respiratory distress syndrome (ARDS), multiple organ failure (MOF), and viral sepsis, are correlated with the cytokine storm. During flu infection, QFPDD reduced the lung indexes and downregulated the expressions of MCP-1, TNF-[Formula: see text], IL-6, and IL-1[Formula: see text] in broncho-alveolar lavage fluid (BALF), lungs, or serum samples. The infiltration of neutrophils and inflammatory monocytes in lungs was decreased dramatically, and lung injury was ameliorated in QFPDD-treated flu mice. In addition, QFPDD also inhibited the polarization of M1 macrophages and downregulated the expressions of IL-6, TNF-[Formula: see text], MIP-2, MCP-1, and IP-10, while also upregulating the IL-10 expression. The phosphorylated TAK1, IKK[Formula: see text]/[Formula: see text], and I[Formula: see text]B[Formula: see text] and the subsequent translocation of phosphorylated p65 into the nuclei were decreased by QFPDD. These findings indicated that QFPDD reduces the intensity of the cytokine storm by inhibiting the NF-[Formula: see text]B signaling pathway during severe viral infections, thereby providing theoretical and experimental support for its clinical application in respiratory viral infections.

Baicalin inhibited both the Furin/TGFß1/Smad3/TSP-1 pathway in endothelial cells and the AKT/Ca2+/ROS pathway in platelets to ameliorate inflammatory coagulopathy.

Inflammatory coagulopathy is resulted from endothelial dysfunction and platelet hyperactivation in inflammatory diseases. In this study, the effects of baicalin, an active component of the traditional Chinese medicine Huangqin, on inflammatory coagulopathy were observed both in vivo and in vitro. In LPS-induced rats, baicalin ameliorated coagulation indexes, inhibited platelet hyperactivation and decreased the expression of thrombospondin-1 (TSP-1) in vessels. In cultured endothelial cells, baicalin decreased the expression of TSP-1 and collagen as well as the TNF-α-induced increase in the levels of TSP-1 and ICAM-1. Baicalin could significantly decrease the platelet adhesion on endothelial cells treated with TNF-α. Baicalin also could inhibit the increase of ROS level and the activation of the NLRP3/Caspase-1/GSDMD pathway in TNF-α-induced endothelial cells. Furin was found to be the direct target of baicalin in HUVECs. Knockdown of Furin using siRNA could ameliorate the effects of baicalin on the activation of TGFß1/Smad3 pathway, TSP-1 expression and the adhesion of platelets on TNF-α-treated endothelial cells. At the same time, baicalin inhibited platelet aggregation induced by collagen or combination of collagen and TSP-1 peptide. Collagen-induced Ca2+ mobilization, ROS level increase, AKT1 phosphorylation, platelet degranulation and TSP-1 release could be all inhibited by baicalin. In all, baicalin ameliorated endothelial dysfunction by inhibiting Furin/TGFß1/Smad3/TSP-1 pathway and also ameliorated platelet activation by inhibiting AKT-related pathway. Both the inhibiting effects of baicalin on endothelial dysfunction and platelet activation might contribute to its ameliorating effects on inflammatory coagulopathy.

[Reduning Injection protects flu-infected mice by inhibiting infiltration of inflammatory cells in lung and down-regulating cytokine storm].

This study aimed to explore the protective effect of Reduning Injection(RDN) on mice infected by influenza virus A/PR/8(PR8) and its immune regulatory roles during viral infection. In in vivo experiments, female C57 BL/6 mice were randomly divided into phosphate buffered saline(PBS) group, PR8-infected group, oseltamivir treatment group(OSV) and RDN treatment group. After 2 h of PR8 infection, mice in the oseltamivir group were gavaged with oseltamivir 30 mg·kg~(-1), and those in the RDN treatment group were injected intraperitoneally with RDN 1.5 mL·kg~(-1)once per day for seven consecutive days. The body weight of mice in each group was recorded at the same time every morning for 16 consecutive days. The line chart of body weight change was created to analyze the protective effect of RDN on flu-infected mice. The relative mRNA expression of different cytokines(IL-6, TNF-α, MCP-1, IL-1ß, MIP-2, IP-10 and IL-10) in lung samples of flu-infected mice was detected by PCR. Flow cytometry was utilized to analyze the composition of immune cells of mouse BALF samples on day 5 after infection. Mouse macrophage cell line RAW264.7 was planted and treated by different concentrations of RDN(150, 300, 600 µg·mL~(-1)) for 24 h or 48 h, and cell proliferation was detected by CCK-8 assay. RAW264.7 cells and mouse primary peritoneal macrophages were stimulated with synthetic single stranded RNA(R837), which elicited the inflammatory response by mimicking the infection of single-stranded RNA viruses. The expression of cytokines and chemokines in the supernatants of above culture system was detected by ELISA and qPCR. On days 4, 5, 6, 7 and 15 after infection, the body weight loss of mice in the RDN treatment group was alleviated compared with that of PR8-infected mice(P<0.05). RDN treatment obviously reduced lung index and the production of IL-6, TNF-α, MCP-1 and MIP-2 in lung tissues of flu-infected mice(P<0.05). The proportions of macrophages, neutrophils and T cells in mouse BALF samples were analyzed by flow cytometry, and compared with PR8-infected mice, RDN decreased the proportion of macrophages in BALF of flu-infected mice(P<0.05), and the proportion of T cells was recovered dramatically(P<0.001). In CCK-8 assay, the concentrations of RDN(150, 300, 600 µg·mL~(-1)) failed to cause cytotoxicity to RAW264.7 cells. In addition, RDN lowered the expression of inflammatory cytokines such as IL-6, TNF-α,MCP-1, IL-1ß, RANTES, and IP-10 and even anti-inflammatory cytokine IL-10 in R837-induced macrophages. RDN reduced the infiltration of inflammatory macrophages and the production of excessive inflammatory cytokines, alleviated the body weight loss of flu-infected mice. What's more, RDN restored the depletion of T cells, which might prevent secondary infection and deteriorative progression of the disease. Taken together, RDN may inhibit cytokine production and therefore down-regulate cytokine storm during the infection of influenza virus.

The licorice flavonoid isoliquiritigenin attenuates Mycobacterium tuberculosis-induced inflammation through Notch1/NF-κB and MAPK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Glycyrrhiza is a small perennial herb that has been traditionally used to treat many diseases across the world. Licorice (Gancao in Chinese) is the dried root and rhizome of G. glabra, G. uralensis or G. inflata. Licorice plays an important role in traditional Chinese medicine (TCM), and is the most frequently used in Chinese herbal formulas. Isoliquiritigenin (ISL) is a flavonoid extracted from licorice, and has been evaluated for its various biological activities, including anti-inflammatory, anti-tumor and anti-oxidant activities. Excessive and persistent inflammation in the Mycobacterium tuberculosis (Mtb) infection is not conducive to the elimination of Mtb, but contributes to serious pulmonary dysfunction. AIM OF THE STUDY: This study aimed to examine the anti-inflammatory effects of ISL in the Mtb infection. METHODS: In vitro models of Mtb-infected macrophages were established. Murine macrophage Raw 264.7 cells and primary peritoneal macrophages were used in this study. Cell viability was determined by the cell counting kit-8 (CCK-8) assay. The effects of ISL on the secretion levels of interleukin -1ß (IL-1ß), tumor necrosis factor -α (TNF-α), and interleukin -6 (IL-6) were detected by the enzyme-linked immunosorbent assay (ELISA). The expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were measured by the real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. Western blot was used to assess the effects of ISL on the activation of NLRP3 inflammasome and Notch1/NF-κB and MAPK signaling pathways. Immunofluorescence assays was used to detected the translocation of phosphorylation of p65 subunit of NF-κB. RESULTS: It was revealed that ISL inhibited the secretion of IL-1ß and the activation of pore-forming protein (gasdermin D, GSDMD) by suppressing the activation of NLPR3 inflammasome induced by Mtb infection. ISL was also shown to have promising inhibitory effects on inflammatory factors, such as TNF-α, IL-6, iNOS and COX2. Regarding the anti-inflammatory mechanism of ISL, it was found that ISL exerted its anti-inflammatory effects by inhibiting the activation of Notch1/NF-κB and MAPK signaling pathways. CONCLUSION: ISL reduced Mtb-induced inflammation through the Notch1/NF-κB and MAPK signaling pathways. ISL might be used as a potential adjuvant drug to treat tuberculosis by adjusting host immune responses.

Quercitrin improved cognitive impairment through inhibiting inflammation induced by microglia in Alzheimer's disease mice.

OBJECTIVE: Diets rich in quercitrin show a neuroprotective effect, but the mechanism is not very clear at present. The objective of this study is to explore the effect and mechanism of quercitrin in the treatment of alzheimer's disease (AD). METHODS: 5XFAD transgenic mice were fed with a diet supplemented with quercitrin for three consecutive months. Behavioral experiments were conducted to assess the cognitive ability, luminex liquid chip technology was used to assess the production of proinflammatory cytokines and immunohistochemistry was used to elucidate the activation of microglia. RESULTS: Quercitrin increased the frequency in exploring new objects, shortened the escape latency and increased the frequency crossing the platform in AD model mice. Quercitrin inhibited the activation and proliferation of microglia, inhibited the secretion of inflammatory cytokines and chemokines and reduced the accumulation of amyloid-ß plaques in AD model mice. CONCLUSION: Quercitrin improved mice cognitive impairment through alleviating the intensity of inflammatory response and is a promising medicinal plant extract in the treatment of AD.

Systems pharmacological study illustrates the immune regulation, anti-infection, anti-inflammation, and multi-organ protection mechanism of Qing-Fei-Pai-Du decoction in the treatment of COVID-19.

BACKGROUND: The traditional Chinese medicine (TCM) formula Qing-Fei-Pai-Du decoction (QFPDD) was the most widely used prescription in China's campaign to contain COVID-19, which has exhibited positive effects. However, the underlying mode of action is largely unknown. PURPOSE: A systems pharmacology strategy was proposed to investigate the mechanisms of QFPDD against COVID-19 from molecule, pathway and network levels. STUDY DESIGN AND METHODS: The systems pharmacological approach consisted of text mining, target prediction, data integration, network study, bioinformatics analysis, molecular docking, and pharmacological validation. Especially, we proposed a scoring method to measure the confidence of targets identified by prediction and text mining, while a novel scheme was used to identify important targets from 4 aspects. RESULTS: 623 high-confidence targets of QFPDD's 12 active compounds were identified, 88 of which were overlapped with genes affected by SARS-CoV-2 infection. These targets were found to be involved in biological processes related with the development of COVID-19, such as pattern recognition receptor signaling, interleukin signaling, cell growth and death, hemostasis, and injuries of the nervous, sensory, circulatory, and digestive systems. Comprehensive network and pathway analysis were used to identify 55 important targets, which regulated 5 functional modules corresponding to QFPDD's effects in immune regulation, anti-infection, anti-inflammation, and multi-organ protection, respectively. Four compounds (baicalin, glycyrrhizic acid, hesperidin, and hyperoside) and 7 targets (AKT1, TNF-α, IL6, PTGS2, HMOX1, IL10, and TP53) were key molecules related to QFPDD's effects. Molecular docking verified that QFPDD's compounds may bind to 6 host proteins that interact with SARS-CoV-2 proteins, further supported the anti-virus effect of QFPDD. At last, in intro experiments validated QFPDD's important effects, including the inhibition of IL6, CCL2, TNF-α, NF-κB, PTGS1/2, CYP1A1, CYP3A4 activity, the up-regulation of IL10 expression, and repressing platelet aggregation. CONCLUSION: This work illustrated that QFPDD could exhibit immune regulation, anti-infection, anti-inflammation, and multi-organ protection. It may strengthen the understanding of QFPDD and facilitate more application of this formula in the campaign to SARS-CoV-2.

Baicalin protects mice from infection with methicillin-resistant Staphylococcus aureus via alleviating inflammatory response.

Sepsis was redefined as life-threatening organ dysfunction caused by a dysregulated host response to infection in 2016. One of its most common causes is Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA), which leads to a significant increase in morbidity and mortality. Therefore, innovative and effective approaches to combat MRSA infection are urgently needed. Recently, host-directed therapy (HDT) has become a new strategy in the treatment of infectious diseases, especially those caused by antibiotic-resistant bacteria. Baicalin (BAI) is the predominant flavonoid and bioactive compound isolated from the roots of Radix Scutellariae (Huang Qin), a kind of traditional Chinese medicine. It has been reported that BAI exhibits multiple biological properties such as anti-oxidant, antitumor, and anti-inflammatory activities. However, the therapeutic role of BAI in MRSA infection is still unknown. In this study, it is found that BAI treatment inhibited the production of IL-6, TNF-α, and other cytokines from MRSA- or bacterial mimics-stimulated Mϕs and dendritic cells (DCs). BAI played an anti-inflammatory role by inhibiting the activation of ERK, JNK MAPK, and NF-κB pathways. Moreover, the serum level of TNF-α was decreased, whereas IL-10 was increased, in mice injected with MRSA. Furthermore, the bacterial load in livers and kidneys were further decreased by the combination of BAI and vancomycin (VAN), which might account for the amelioration of tissue damage. BAI reduced the high mortality rate caused by MRSA infection. Collectively, the results suggested that BAI may be a viable candidate of HDT strategy against severe sepsis caused by antibiotic-resistant bacteria such as MRSA.

G protein-coupled estrogen receptor, a potential therapeutic target of asthma probed by Chinese herb.

Discussion on the identification of GPER as a potential therapeutic target for asthma through Chinese herb-driven drug discovery strategy.

Improvement cues of lesion absorption using the adjuvant therapy of traditional Chinese medicine Qinbudan tablet for retreatment pulmonary tuberculosis with standard anti-tuberculosis regimen.

BACKGROUND: China is the second highest pulmonary tuberculosis (PTB) burden country worldwide. However, retreatment of PTB has often developed resistance to at least one of the four first-line anti-TB drugs. The cure rate (approximately 50.0-73.3%) and management of retreatment of PTB in China needs to be improved. Qinbudan decoction has been widely used to treat PTB in China since the 1960s. Previously clinical studies have shown that the Qinbudan tablet (QBDT) promoted sputum-culture negative conversion and lesion absorption. However, powerful evidence from a randomized controlled clinical trial is lacking. Therefore, the aim of this study was to compare the efficacy and safety of QBDT as an adjunct therapy for retreatment of PTB. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled clinical trial in China. People diagnosed with PTB were enrolled who received previous anti-TB treatment from April 2011 to March 2013. The treatment group received an anti-TB regimen and QBDT, and the control group was administered an anti-TB regimen plus placebo. Anti-TB treatment options included isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin for 2 months (2HRZES), followed by isoniazid, rifampicin, ethambutol for 6 months (6HRE), daily for 8 months. Primary outcome was sputum-culture conversion using the MGIT 960 liquid medium method. Secondary outcomes included lung lesion absorption and cavity closure. Adverse events and reactions were observed after treatment. A structured questionnaire was used to record demographic information and clinical symptoms of all subjects. Data analysis was performed by SPSS 25.0 software in the full analysis set (FAS) population. RESULTS: One hundred eighty-one cases of retreatment PTB were randomly divided into two groups: the placebo group (88 cases) and the QBDT group (93 cases). A total of 166 patients completed the trial and 15 patients lost to follow-up. The culture conversion rate of the QBDT group and placebo group did not show a noticeable improvement by using the covariate sites to correct the rate differences (79.6% vs 69.3%; rate difference = 0.10, 95% confidence interval (CI): - 0.02-0.23; F = 2.48, P = 0.12) after treatment. A significant 16.6% increase in lesion absorption was observed in the QBDT group when compared with the placebo group (67.7% vs 51.1%; rate difference = 0.17, 95% CI: 0.02-0.31; χ2 = 5.56, P = 0.02). The intervention and placebo group did not differ in terms of cavity closure (25.5% vs 21.1%; rate difference = 0.04, 95% CI: - 0.21-0.12; χ2 = 0.27, P = 0.60). Two patients who received chemotherapy and combined QBDT reported pruritus/nausea and vomiting. CONCLUSIONS: No significant improvement in culture conversion was observed for retreatment PTB with traditional Chinese medicine plus standard anti-TB regimen. However, QBDT as an adjunct therapy significantly promoted lesion absorption, thereby reducing lung injury due to Mycobacterium tuberculosis infection. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov, NCT02313610.

Xuebijing Injection Alleviates Pam3CSK4-Induced Inflammatory Response and Protects Mice From Sepsis Caused by Methicillin-Resistant Staphylococcus aureus.

A leading cause of death worldwide is sepsis that develops as a dysregulated immune response to infection. Serious infection caused by methicillin-resistant Staphylococcus aureus (MRSA) increases the difficulty of treatment in septic patients. Host-directed therapy (HDT) is an emerging approach to bacterial infections. Xuebijing injection (XBJ), a commercialized injectable prescription from traditional Chinese medicine, has been used as adjuvant therapy for sepsis with a history of 15 years. Whether it plays a protective role in severe infection caused by antibiotic-resistant bacteria is still unknown. In this study, XBJ significantly improved the survival of MRSA-induced sepsis mice. In MRSA-infected mouse model, XBJ down-regulated the expression of inflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, MCP-1, MIP-2, and IL-10 in sera. Besides that, it decreased the bacterial load in spleens, livers, and alleviated tissue damage of lung, liver, and kidney. The combination of XBJ with vancomycin or dexamethasone exhibited a better down-regulatory role of the inflammatory response. Then, the protective mechanism of XBJ was further investigated. XBJ inhibited heat-killed MRSA-induced IL-6 and TNF-α production in mouse macrophages. XBJ also decreased Pam3CSK4 (a synthetic tripalmitoylated lipopeptide mimicking bacterial lipoproteins)-stimulated expression of IL-6, TNF-α, IL-1ß, IL-12, etc. in mouse macrophages. Furthermore, XBJ down-regulated the activation of NF-κB, MAPK, and PI3K/Akt pathways in Pam3CSK4-stimulated mouse macrophages. In conclusion, our findings demonstrated that XBJ played a protective role in MRSA-challenged mice and down-regulated the inflammatory response and the activation of signaling pathways initiated by Pam3CSK4. It enlarged the clinical application of XBJ in the treatment of severe bacterial infection, e.g. caused by MRSA.

Secoeudesma sesquiterpenes lactone A alleviates inflammation and offers adjuvant protection in severe infection of carbapenem-resistant Klebsiella pneumoniae.

ETHNOPHARMACOLOGICAL RELEVANCE: Secoeudesma sesquiterpenes lactone A (SESLA) is a sesquiterpene compound isolated from Inula japonica Thunb. (I. japonica). It is an herb widely distributed in Asian countries often used for the treatment of various conditions including tumors, bronchitis and bacterial and viral infections. It has been reported that SESLA could significantly inhibit the production of nitric oxide (NO) by lipopolysaccharide (LPS) in Raw264.7 cells. However, the mechanism responsible for this anti-inflammatory role and its role in the treatment of antibiotic-resistant bacterial infection, e.g., carbapenem-resistant Klebsiella pneumoniae (CRKP), remain to be investigated. AIM OF THE STUDY: This study was carried out to investigate the protective anti-inflammatory role and the underlying molecular mechanisms of SESLA in LPS or CRKP evoked inflammation. MATERIALS AND METHODS: ELISA and PCR were utilized to detect the expression of inflammatory mediators in LPS or heat-killed CRKP (HK CRKP)-stimulated immune cells containing different concentrations of SESLA. The protective role of SESLA was observed in mice challenged with a lethal dose of CRKP. Mice were intraperitoneally injected with CRKP to create a septic mouse model to evaluate the protective role of SESLA in vivo. Phosphorylated proteins, which represented the activation of signaling pathways, were examined by Western blot. RESULTS: SESLA was showed to inhibit the expression of inflammatory mediators in various macrophages and dendritic cells upon stimulation of LPS or HK CRKP. It also facilitated phagocytosis of bacteria by Raw264.7 cells. The combined use of SELSA and the ineffective antibiotic, meropenem, increased the survival rate of CRKP infected mice from 25% to 50%. ERK, NF-κB and PI3K/Akt pathways accounted for the anti-inflammatory role of SESLA with the stimulation of LPS. CONCLUSION: According to the notable anti-inflammatory effect in vitro and its joint protective effects on a septic mouse model, SESLA might act as an adjuvant drug candidate for sepsis, even those caused by antibiotic-resistant bacteria, e.g., CRKP.

Portulaca Extract Attenuates Development of Dextran Sulfate Sodium Induced Colitis in Mice through Activation of PPARγ.

Portulaca oleracea L. is a traditional Chinese medicine, which has been used as adjuvant therapy for inflammatory bowel disease (IBD). However, the mechanism of its activity in IBD still remains unclear. Since previous studies have documented the anti-inflammatory effect of peroxisome proliferator activated receptors-γ (PPAR-γ), Portulaca regulation of PPAR-γ in inflammation was examined in current study. Ulcerative colitis (UC) was generated by 5% dextran sulfate sodium (DSS) in mice and four groups were established as normal control, DSS alone, DSS plus mesalamine, and DSS plus Portulaca. Severity of UC was evaluated by body weight, stool blood form, and length of colorectum. Inflammation was examined by determination of inflammatory cytokines (TNF-a, IL-6, and IL-1a). Portulaca extract was able to attenuate development of UC in DSS model similar to the treatment of mesalazine. Moreover, Portulaca extract inhibited proinflammatory cytokines release and reduced the level of DSS-induced NF-κB phosphorylation. Furthermore, Portulaca extract restored PPAR-γ level, which was reduced by DSS. In addition, Portulaca extract protected DSS induced apoptosis in mice. In conclusion, Portulaca extract can alleviate colitis in mice through regulation of inflammatory reaction, apoptosis, and PPAR-γ level; therefore, Portulaca extract can be a potential candidate for the treatment of IBD.

Antimycobacterial and Anti-inflammatory Mechanisms of Baicalin via Induced Autophagy in Macrophages Infected with Mycobacterium tuberculosis.

Tuberculosis (TB) remains a leading killer worldwide among infectious diseases and the effective control of TB is still challenging. Autophagy is an intracellular self-digestion process which has been increasingly recognized as a major host immune defense mechanism against intracellular microorganisms like Mycobacterium tuberculosis (Mtb) and serves as a key negative regulator of inflammation. Clinically, chronic inflammation surrounding Mtb can persist for decades leading to lung injury that can remain even after successful treatment. Adjunct host-directed therapy (HDT) based on both antimycobacterial and anti-inflammatory interventions could be exploited to improve treatment efficacy and outcome. Autophagy occurring in the host macrophages represents a logical host target. Here, we show that herbal medicine, baicalin, could induce autophagy in macrophage cell line Raw264.7 and caused increased killing of intracellular Mtb. Further, baicalin inhibited Mtb-induced NLRP3 inflammasome activation and subsequent inflammasome-derived IL-1ß. To investigate the molecular mechanisms of baicalin, the signaling pathways associated with autophagy were examined. Results indicated that baicalin decreased the levels of phosphorylated protein kinase B (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) at Ser473 and Ser2448, respectively, but did not alter the phosphorylation of p38, JNK, or ERK both in Raw264.7 and primary peritoneal macrophages. Moreover, baicalin exerted an obvious inhibitory effect on nuclear factor-kappa B (NF-κB) activity. Finally, immunofluorescence studies demonstrated that baicalin promoted the co-localization of inflammasome with autophagosome may serve as the underlying mechanism of autophagic degradative effect on reducing inflammasome activation. Together, baicalin definitely induces the activation of autophagy on the Mtb-infected macrophages through PI3K/Akt/mTOR pathway instead of MAPK pathway. Furthermore, baicalin inhibited the PI3K/Akt/NF-κB signal pathway, and both autophagy induction and NF-κB inhibition contribute to limiting the NLRP3 inflammasome as well as subsequent production of pro-inflammatory cytokine IL-1ß. Based on these results, we conclude that baicalin is a promising antimycobacterial and anti-inflammatory agent which can be a novel candidate for the development of new adjunct drugs targeting HDT for possible improved treatment.

MCL Plays an Anti-Inflammatory Role in Mycobacterium tuberculosis-Induced Immune Response by Inhibiting NF-κB and NLRP3 Inflammasome Activation.

Mycobacterium tuberculosis (Mtb) remains a significant menace to global health as it induces granulomatous lung lesions and systemic inflammatory responses during active tuberculosis (TB). Micheliolide (MCL), a sesquiterpene lactone, was recently reported to have a function of relieving LPS-induced inflammatory response, but the regulative role of MCL on the immunopathology of TB still remains unknown. In this experiment, we examined the inhibitory effect of MCL on Mtb-induced inflammatory response in mouse macrophage-like cell line Raw264.7 by downregulating the activation of nuclear factor kappa B (NF-κB) and NLRP3 inflammasome. Evidences showed that MCL decreased the secretion of Mtb-induced inflammatory cytokines (IL-1ß and TNF-α) in a dose-dependent manner. Meanwhile, MCL dramatically suppressed Mtb-induced activation of iNOS and COX2 as well as subsequent production of NO. Furthermore, MCL inhibited Mtb-induced phosphorylation of Akt (Ser 473) in Raw264.7. According to our results, MCL plays an important role in modulating Mtb-induced inflammatory response through PI3K/Akt/NF-κB pathway and subsequently downregulating the activation of NLRP3 inflammasome. Therefore, MCL may represent as a potential drug candidate in the adjuvant treatment of TB by regulating host immune response.

Identification of plant-derived natural products as potential inhibitors of the Mycobacterium tuberculosis proteasome.

BACKGROUND: The Mycobacterium tuberculosis (Mtb) proteasome has been established as a viable target for the development of anti-tuberculosis agents. In this study, the inhibitory activities of 100 plant-derived natural products on the Mtb proteasome were analyzed to identify novel potential inhibitors. METHODS: The fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC can be hydrolyzed by the proteasome to release free AMC, the fluorescence of which is proportional to the proteasome activity. The inhibitory activities of 100 natural products (each at a final concentration of 200 µM) were detected by this method using MG132 as a positive control. RESULTS: Twelve of these natural products (10 of which were flavonoids) inhibited the activity of the Mtb proteasome by more than 65%. Comparison of the structural differences between the flavonoids with good inhibitory activity and those without inhibitory activity revealed that the hydroxyl at the flavonoid C ring C-3 or the hydroxyl/methoxyl at the flavonoid A ring C-6 were critical for the inhibition of proteasomal activity. CONCLUSIONS: These data indicate that flavonoids represent a basis for rational structural design in the process of novel anti-tuberculosis drug discovery.