Preservation of mitochondrial homeostasis is responsible for the ameliorative effects of Suhuang antitussive capsule on non-resolving inflammation via inhibition of NF-κB signaling and NLRP3 inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Suhuang antitussive capsule (Suhuang), one of traditional antitussive Chinese patent medicines, has been used for the treatment of post-infectious cough and cough variant asthma in clinical practice. It has been demonstrated to show numerous biological actions including antitussive and anti-inflammatory effects. AIM OF THE STUDY: This study aims to investigate the effects of Suhuang on non-resolving inflammation and its underlying molecular mechanism. MATERIAL AND METHODS: In vitro, mitochondrial membrane potential and ROS were detected by flow cytometry analysis. mtDNA release and mPTP fluorescence were determined by Q-PCR and fluorescence microplate reader analysis. Cytochrome C release and 8-OHdG levels were evaluated by ELISA. Additionally, the effects of Suhuang on Drp1, MMP9, IκBα/NF-κB and NLRP3/ASC/Caspase-1 expression were determined by Q-PCR, gelatin zymography or immunoblot analysis. In vivo, C57/BL6 mice were orally administrated for 2 weeks with Suhuang, then lung injury was induced by LPS. Inflammatory mediators mRNA, histological assessment and NF-κB/Caspase-1/IL-1ß levels were evaluated by Q-PCR, H&E staining and immunoblot analysis. Two sepsis models of mice were further used to evaluate its anti-inflammatory effects. RESULTS: Suhuang restored mitochondrial homeostasis by inhibiting Drp1 activation and mitochondrial fission. Besides, Suhuang reduced mPTP opening, mitochondrial membrane potential collapse, ROS overproduction and mtDNA release. Moreover, Suhuang down-regulated MMP9 expression. As a consequence of preserved mitochondrial homeostasis, Suhuang inhibited NF-κB pathway activation by prevention of NF-κB-p65 phosphorylation and IκBα degradation. Suhuang also limited NLRP3 inflammasome activation by blocking NLRP3-ASC interaction and promoting NLRP3 ubiquitination degradation. Drp1 knockdown in vitro diminished the inhibitory effects of Suhuang on inflammatory responses, indicating the essential role of Drp1 in the Suhuang's activity. Consistently, the therapeutic effects of Suhuang were confirmed in LPS-inhaled mice, which recapitulated the protective actions of Suhuang in mitochondrial homeostasis in vitro. Additionally, two sepsis models of mice confirmed the inhibitory effects of Suhuang on uncontrolled inflammation. CONCLUSIONS: Altogether, our work reveals that Suhuang inhibits non-resolving inflammation through inhibition of NF-κB signaling and NLRP3 inflammasome activation by preserving mitochondrial homeostasis, providing new pharmacological data for the clinical use of Suhuang. Our study also suggests mitochondrial homeostasis as a potential intrinsic regulatory strategy for treating inflammatory diseases.

A new target for the treatment of inflammatory bowel disease: Interleukin-37.

Interleukin (IL)-37 belongs to the IL-1 cytokine family. It has anti-inflammatory effects on numerous autoimmune diseases such as asthma, psoriasis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and rheumatoid arthritis (RA). Mechanistically, IL-37 plays an anti-inflammatory role by regulating the expression of inflammatory factors in two ways: binding extracellular receptors IL-18R or transferring into the nucleus with Smad3. IBD is a kind of idiopathic intestinal inflammatory disease with unknown etiology and pathogenesis. Recent researches had proved that IL-37 is negatively involved in the pathogenesis and development of IBD. Among various inflammatory diseases, IL-37 has been shown to regulate inflammatory development by acting on various immune cells such as neutrophils, macrophages (Mϕ), dendritic cells (DCs), T cells and intestinal epithelial cells. This review summarizes the biological role of IL-37, and its immunoregulatory effects on the immune cells, especially anti-inflammatory function in both human and experimental models of IBD.

Identification of quality control markers in Suhuang antitussive capsule based on HPLC-PDA fingerprint and anti-inflammatory screening.

Suhuang antitussive capsule (SH), one of traditional Chinese patent medicines, has been widely used for treating cough variant asthma and postinfectious cough in clinic. The objective of this work is to identify the characteristic and active ingredients as the quality control markers for SH based on high performance liquid chromatography with photodiode array detector (HPLC-PDA) fingerprint and screening of anti-inflammatory components. Similarity analysis (SA), hierarchical clustering analysis (HCA) and principal component analysis (PCA) were used to evaluate 16 different batches of SH. 13 compounds accounting for 36% of the total components in the fingerprint were identified and semi-quantitatively analyzed, which anti-inflammatory activity was tested with the in vitro assay. The results showed that the established chemical fingerprint could clearly distinguish different batches of SH by SA, HCA, and PCA analysis. Furthermore, four known compounds (chlorogenic acid, schisandrin, angeloylgomisin H and praeruptorin A) were screened out to be the most discriminant variables, which could be applied to quality control of SH by quantitative analysis. The semi-quantitative results showed that six compounds were major components, i.e. arctiin (10.28 ±â€¯3.18 mg/g), ephedrine (9.26 ±â€¯1.58 mg/g), schisandrin (3.09 ±â€¯0.83 mg/g), pseudoephedrine (2.34 ±â€¯1.04 mg/g), schisandrin B (1.48 ±â€¯0.16 mg/g), and 1-caffeoylquinic acid (1.36 ±â€¯0.42 mg/g). The anti-inflammatory results showed that SH extract, praeruptorin A, schisandrin, arctigenin and pseudoephedrine could significantly inhibit inflammatory mediator NO production in LPS-stimulated RAW264.7 macrophages. These findings indicated that praeruptorin A, schisandrin, arctiin and pseudoephedrine could be proposed as the quality control markers for SH.

Suhuang antitussive capsule inhibits NLRP3 inflammasome activation and ameliorates pulmonary dysfunction via suppression of endoplasmic reticulum stress in cough variant asthma.

Pulmonary dysfunction is tightly associated with cough variant asthma (CVA), a respiratory damage disease. Suhuang antitussive capsule (Suhuang), one of traditional Chinese patent medicines, plays a crucial role in the treatment and complication of CVA in the long clinical application. In this study, we aimed to investigate the protective effects and underlying antitussive mechanisms of Suhuang on pulmonary function in ovalbumin (OVA)-induced CVA rats. Administration (i.g.) of Suhuang significantly alleviated pulmonary damage and dysfunction. Suhuang improved ER stress and PKCε translocation via regulation of Ca2+ trafficking. Suhuang also inhibited NLRP3 inflammasome activation, as evidenced by disrupting the assembly of NLRP3 inflammasome and reducing the expression of cleaved caspase-1, and decreased IL-1ß secretion. Besides, it's identified that TXNIP induction and RIP1-RIP3-Drp1 pathway were required for the inhibitory routes of Suhuang from ER stress to NLRP3 inflammasome activation. Consistent with the in vivo findings, Suhuang also attenuated ER stress/NLRP3 inflammasome activation, and thereby restored pulmonary homeostasis in vitro. Meantime, these functions were diminished by blocking ER stress, indicating that ER stress is essential for the effects of Suhuang on pulmonary function. A further in vivo analysis showed that Suhuang-driven pharmacological inactivation of NLRP3 inflammasome and amelioration of pulmonary dysfunction were reversed by an ER stress inducer tunicamycin, well confirming the beneficial effects of Suhuang on pulmonary function by regulation of ER stress. Collectively, these results indicated that Suhuang contributed to impairing NLRP3 inflammasome activation via inhibition of ER stress, which was responsible for the protection of pulmonary homeostasis. These findings may provide a pharmacological groundwork and important new experimental data regarding the clinical treatment of Suhuang in CVA patients.

[A new phenolic glycoside from honey-fried Eriobotrya japonica].

A total of twelve compounds were isolated from the ethyl acetate of the water extract of honey-fried Eriobotrya japonica through column chromatography over silica gel,Sephadex LH-20,RP-18,and preparative HPLC. Their structures were established by MS,1 D NMR and 2 D NMR data as japonicanoside A( 1),nerolidol-3-O-α-L-rhamnopyranosyl-( 1→2)-ß-D-glucopyranoside( 2),nerolidol-3-O-α-L-rhamnopyranosyl-( l→4)-α-L-rhamnopyranosyl-( 1 → 2)-[α-L-( 4-trans-feruloyl)-rhamnopyranosyl-( 1 → 6) ]-ß-D-glucopyranoside( 3),( +)-catechin( 4),(-)-epicatechin( 5),kaempferol 3-O-α-L-rhamnopyranoside( 6),quercitrin( 7),quercetin-3-O-ß-D-galactopyranoside( 8),quercetin-3-O-ß-glucopyranoside( 9),vanillin( 10),protocatechuic aldehyde( 11),and maltol( 12). Among them,1 is a new phenolic glycoside.

Suhuang Antitussive Capsules-Ameliorative Effects on LPS-Induced Sputum Obstruction in Mice Through Promoting HGF Secretion.

Sputum obstruction is one of common cough complications, which is tightly associated with airway inflammation. Suhuang antitussive capsule (SH Capsule), a classic traditional Chinese medicine prescription, has been used for the treatment of post-cold cough and cough variant asthma in the long clinical application. This study aims to investigate the effects and underlying mechanisms of SH Capsule on LPS-induced sputum obstruction in mice. The results showed that SH Capsule effectively promoted the tracheal phenol red output and mucociliary clearance. SH Capsule also alleviated airway inflammation-mediated mucin 5AC (MUC5AC) level through EGFR-ERK signaling. A further in vivo analysis showed that HGF inhibitor SU11274 abrogated the effects of SH Capsule on MUC5AC, well demonstrating that HGF was required for the beneficial effects of SH Capsule on expectoration in vivo. Moreover, SH Capsule promoted HGF secretion in a colon-dependent manner, which reached lung tissues via blood circulation. Collectively, this study provided new pharmacological data for clinical use of SH Capsule, and proposed a novel mechanism by which SH Capsule was pharmacologically promising for treating sputum obstruction.

Generation of Induced Cardiospheres via Reprogramming of Mouse Skin Fibroblasts.

Cardiospheres represent a more effective cell-based therapy for treatment of myocardial infarction than stem cells of non-cardiac origin. Unfortunately, their therapeutic application is limited by low yield of cell harvesting, declining quality and quantity during the aging process, and the need for highly invasive heart biopsy. Therefore, there is an emerging interest in generating cardiosphere-like stem cells from somatic cells via somatic reprogramming. This novel approach would provide an unlimited source of stem cells with cardiac differentiation potential. Here we provide the detailed protocol for generating induced cardiospheres (iCS) for cardiac regeneration by somatic reprogramming of mouse fibroblasts using a panel of pluripotent transcription factors and cardiotrophic growth factors. © 2018 by John Wiley & Sons, Inc.

Optimized Plasmid Construction Strategy for Cas9.

BACKGROUND/AIMS: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). METHODS: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. RESULTS: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. CONCLUSIONS: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.

Optimized guide RNA structure for genome editing via Cas9.

The genome editing tool Cas9-gRNA (guide RNA) has been successfully applied in different cell types and organisms with high efficiency. However, more efforts need to be made to enhance both efficiency and specificity. In the current study, we optimized the guide RNA structure of Streptococcus pyogenes CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system to improve its genome editing efficiency. Comparing with the original functional structure of guide RNA, which is composed of crRNA and tracrRNA, the widely used chimeric gRNA has shorter crRNA and tracrRNA sequence. The deleted RNA sequence could form extra loop structure, which might enhance the stability of the guide RNA structure and subsequently the genome editing efficiency. Thus the genome editing efficiency of different forms of guide RNA was tested. And we found that the chimeric structure of gRNA with original full length of crRNA and tracrRNA showed higher genome editing efficiency than the conventional chimeric structure or other types of gRNA we tested. Therefore our data here uncovered the new type of gRNA structure with higher genome editing efficiency.

Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights.

Bacillus subtilis was engineered into an efficient hyaluronic acid (HA) producer by introducing two inducible artificial operons carrying HA synthase gene from Pasteurella multocida and precursor genes encoding enzymes involved in synthesis of the sugar precursors. A two-stage induction strategy was established for metabolic engineering of recombinant B. subtilis to efficiently produce uniform HA with controlled molecular weights. Strain TPG223 produced larger HA molecules (yield=6.8 g/L; molecular weight=4.5 MDa) than strain PG6181 (yield=2.4 g/L; molecular weight=13 KDa), indicating that the enzymes involved in the synthesis of UDP-glucuronic acid are essential for HA biosynthesis. Strain TPG223 was able to synthesize HA molecules ranging in molecular weight from 8 KDa to 5.4 MDa indicating that size control is achievable in vivo through appropriate tools. The work reported here not only advanced mechanisms research of size control in vivo, but also could be an attractive alternative for commercial preparation of uniform size-defined HA.