Xijiao Dihuang decoction combined with Yinqiao powder promotes autophagy-dependent ROS decrease to inhibit ROS/NLRP3/pyroptosis regulation axis in influenza virus infection.

BACKGROUND: Influenza viral pneumonia is a common complication after influenza virus infection. Xijiao Dihuang Decoction combined with Yinqiao Powder (XDY) is effective on improving influenza viral pneumonia. PURPOSE: This study further explores the anti-inflammatory mechanism of XDY in the treatment of influenza viral pneumonia. STUDY DESIGN: The effects of XDY on inflammation, autophagy, NACHT-LRR-PYD-containing protein 3 (NLRP3) inflammasome and pyroptosis were assessed in the mice with influenza viral pneumonia. In addition, the mouse macrophage cell line (J774A.1) infected with influenza virus was adopted to decode the in vitro effects of XDY on autophagy, reactive oxygen species (ROS), NLRP3 inflammasome and pyroptosis. We analyzed the XDY-induced autophagy, especially the mitophagy-related ROS clearance, and the subsequent inhibition of ROS/NLRP3 inflammasome/pyroptosis signaling in the infected macrophages by different assays based on quantitative polymerase chain reaction, western blot, flow cytometry, immunofluorescence and enzyme-linked immunosorbent assay. RESULTS: In vivo, XDY could effectively improve the lung inflammatory response in the mice with influenza virus pneumonia, due to an intact autophagy flux-promoting effect and the inhibiting roles on NLRP3 inflammasome and pyroptosis. Notably, in vitro, compared with the infected macrophages treated by the NLRP3 inflammasome agonist (Monosodium urate) or the mitochondrial-targeted antioxidant agent, the XDY-dependent treating could inhibit pyroptosis by negatively regulating the signaling axis of ROS/NLRP3 inflammasome/pyroptosis in the influenza virus-infected macrophages. More interestingly, XDY could promote an intact autophagy flux, inducing mitophagy eliminating the damaged mitochondria to reduce the intracellular ROS accumulation, and thus decrease the oxidative stress in the infected macrophages. Especially, the inhibitor of autophagy inition, 3-Methyladenine, could reverse the inhibitory effect of XDY on ROS-NLRP3 inflammasome-mediated pyroptosis, indicating an XDY-promoted mitophagy-dependent ROS scavenging. CONCLUSION: XDY can promote an intact autophagy flux to eliminate damaged mitochondria, namely mitophagy, which reduces the intracellular ROS accumulation contributing to NLRP3 inflammasome activation, restricting pyroptosis and eventually alleviating the influenza virus-induced inflammatory lesions. The obtained results provide new insights into the mechanism of action of XDY in alleviating influenza virus pneumonia, especially the roles of XDY in anti-oxidation, anti-inflammation and anti-pyroptosis, with potential therapeutic targets for future application in integrative medicine.

Rapid qualitative profiling and quantitative analysis of Juglandis Mandshuricae Cortex and seven flavonoids by ultra-high performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry.

Juglandis Mandshuricae Cortex is the bark of Juglans mandshurica Maxim., which has been used as a folk medicine plant in China and India. In this study, an ultra-high performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry method was developed to clarify and quantify the chemical profiling of Juglandis Mandshuricae Cortex rapidly. A total of 113 compounds were characterized. Among them, seven flavonoids were simultaneously quantified in 15 min, including myricetin, myricetrin, taxifolin, kaempferol, quercetin, quercitrin, and naringenin. The method was validated for accuracy, precision, and the limits of detection and quantification. All calibration curves showed a good linear relationship (r > 0.9990) within test ranges. The intra- and inter-day relative standard deviations were less than 2.16%. Accuracy validation showed that the recovery was between 95.6 and 101.3% with relative standard deviation values below 2.85%. The validated method was successfully applied to determine the contents of seven flavones in Juglandis Mandshuricae Cortex from seven sources and the contents of these places were calculated respectively. This method provides a theoretical basis for further developing the medicinal value of Juglandis Mandshuricae Cortex.

Uncovering the pharmacological mechanisms of Xijiao Dihuang decoction combined with Yinqiao powder in treating influenza viral pneumonia by an integrative pharmacology strategy.

Xijiao Dihuang decoction combined with Yinqiao powder (XDD-YQP) is a classical combination formula; however, its therapeutic effects in treating influenza viral pneumonia and the pharmacological mechanisms remain unclear. The therapeutic effect of XDD-YQP in influenza viral pneumonia was evaluated in mice. Subsequently, an everted gut sac model coupled with UPLC/Q-TOF MS were used to screen and identify the active compounds of XDD-YQP. Furthermore, network pharmacological analysis was adopted to probe the mechanisms of the active compounds. Lastly, we verified the targets predicted from network pharmacological analysis by differential bioinformatics analysis. Animal experiments showed that XDD-YQP has a therapeutic effect on influenza viral pneumonia. Moreover, 113 active compounds were identified from intestinal absorbed solutions of XDD-YQP. Using network pharmacological analysis, 90 major targets were selected as critical in the treatment of influenza viral pneumonia through 12 relevant pathways. Importantly, the MAPK signaling pathway was found to be closely associated with the other 11 pathways. Moreover, seven key targets, EGFR, FOS, MAPK1, MAP2K1, HRAS, NRAS, and RELA, which are common targets in the MAPK signaling pathway, were investigated. These seven key targets were identified as differentially expressed genes (DEGs) between influenza virus-infected and uninfected individuals. Hence, the seven key targets in the MAPK signaling pathway may play a vital role in the treatment of influenza viral pneumonia with XDD-YQP. This research may offer an integrative pharmacology strategy to clarify the pharmacological mechanisms of traditional Chinese medicines. The results provide a theoretical basis for a broader clinical application of XDD-YQP.

Integrated UPLC-MS/MS and UHPLC-Q-orbitrap HRMS Analysis to Reveal Pharmacokinetics and Metabolism of Five Terpenoids from Alpiniae oxyphyllae Fructus in Rats.

BACKGROUND: Alpiniae oxyphyllae Fructus (AOF), a traditional Chinese medicine (TCM), is widely used in the treatment of urinary, gastrointestinal and neurologic diseases in China. Although terpenoids are the main active ingredients of AOF, there are few researches on their pharmacokinetics and metabolism. METHODS: In this study, a sensitive, rapid, accurate and novel ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to evaluate the pharmacokinetic behavior of five terpenoids (oxyphyllenodiol B, (4S*,5E,10R*)-7-oxo-tri-nor-eudesm-5-en-4ß-ol, 7-epi-teucrenone, (+)- (4R,5S,7R)-13-hydroxynootkatone, (E)-labda-12,14-dien-15(16)-olide-17-oic acid) in rats after oral administration of AOF extracts. 27 metabolic metabolites of the five terpenoids were identified by ultra high performance liquid chromatography -Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) based on precise mass and fragment ions. RESULTS: The established pharmacokinetic analysis method showed good linearity over a wide concentration range, and the lower quantitative limit (LLOQ) ranged from 0.97 to 4.25 ng/mL. Other validation parameters were within the acceptable range. In addition, 27 metabolites were identified in plasma, urine and feces samples, and the metabolic pathways of five terpenoids were mainly focused on glucoside conjugation, dehydration, desaturation and glycine conjugation. CONCLUSION: This is the first study on the pharmacokinetics and metabolism of five terpenoids in AOF, illuminating the disposal process of terpenoids in vivo. It was expected that the results of this study would provide some references for the apprehension of the action mechanism and the further pharmacological study of five terpenoids in AOF.

Effects and safety of tanreqing injection on viral pneumonia: A protocol for systematic review and meta-analysis.

BACKGROUND: Viral pneumonia is a common respiratory disease that leads to high mortality around the world. Tanreqing (TRQ) injection has been widely used to treat viral pneumonia in China. However, the efficiency and safety of TRQ injection for viral pneumonia have not been scientifically and methodically evaluated up to now. Thus, this protocol describes a plan of performing a systematic review and meta-analysis to evaluate the efficacy and safety of TRQ injection on patients with viral pneumonia. METHODS: Only randomized controlled trials will be enrolled in our study, and we will search eligible studies in the following electronic databases: PubMed, Embase, Cochrane Central Register of Controlled Trials, Clinical Trials, China National Knowledge Infrastructure, the Wanfang database, the Chinese Scientific Journal Database, and the Sinomed. The total effective rate of clinical efficacy will be used as primary outcome. Time to relieve symptoms, incidence of adverse reactions, and the laboratory parameters will be used as secondary outcomes. Any side effects and adverse events will be recorded and assessed as safety outcomes. Study inclusion, data extraction, and quality assessment will be performed independently by 2 reviewers, and any disagreement will be resolved by a third reviewer. After that, data synthesis and subgroup analysis will be conducted with the Review Manager V.5.3.3 software. RESULTS: This review will provide a high-quality synthesis to assess the effectiveness and safety of TRQ injection for viral pneumonia patients. CONCLUSION: Our study will provide comprehensive evidence to decide whether TRQ injection is effective and safe for viral pneumonia patients. PROSPERO REGISTRATION NUMBER: PROSPERO CRD42020164164.

[Isolation of endophytic fungi from Macleaya cordata and screening of sanguinarine-producing strains].

Endophytic fungi were isolated from Macleaya cordata growing in Dabie Mountain by agar-block method, and then the endophytic fungi were grouped into different types based on their morphological characteristics, and thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were employed to determine whether the metabolic substances contained sanguinarine or not, and then preliminarily identified by morphological method. The results showed that the leaves hosted the largest number of endophytes (96 isolates) followed by the stems (57 isolates) and finally the roots (28 isolates), respectively. Based on morphological characteristics the endophytic fungi were grouped into 26 types in our study. TLC and HPLC results showed that there was sanguinarine in the metabolic substances of BLH 51 strain. According to the morphological characteristic, the BLH 51 strain was identified as Fusarium proliferatum. All these indicated that the medicinal plant M. cordata harbors abundant endophytes, which could be a new source for the search of active secondary metabolites.