Baicalin relieves complement alternative pathway activation-induced lung inflammation through inhibition of NF-κB pathway.

INTRODUCTION: Acute lung injury (ALI) as one kind of acute pulmonary inflammatory disorder, manifests primarily as damage to alveolar epithelial cells and microvascular endothelial cells. Activation of the complement system is a common pathological mechanism in ALI induced by diverse factors, with the complement alternative pathway assuming a pivotal role. Baicalin, a flavonoid derived from the root of Scutellaria baicalensis Georgi, exhibits noteworthy biological activities. The present study attempted the interventional effects and underlying mechanisms of baicalin in microangiopathy in ALI induced by complement alternative pathway activation. METHODS: Activation of the complement alternative pathway by cobra venom factor (CVF). HMEC cells were pretreated with baicalin and then exposed to complement activation products. The expression of inflammatory mediators was detected by ELISA, and the intranuclear transcriptional activity of NF-κB was assessed by a dual fluorescent kinase reporter gene assay kit. Before establishing the ALI mouse model, baicalin or PDTC was gavaged for 7 d. CVF was injected into the tail vein to establish the ALI model. The levels of inflammatory mediators in BALF and serum were determined by ELISA. HE staining and immunohistochemistry evaluated pathological changes, complement activation product deposition, and NF-κB p65 phosphorylation in lung tissue. RESULTS: Baicalin reduced complement alternative activation product-induced expression of HMEC cells adhesion molecules (ICAM-1, VCAM-1, E-selectin) and cytokines (IL-6, TNF-α) as well as upregulation of NF-κB intranuclear transcriptional activity. Baicalin intervention reduced the number of inflammatory cells and protein content in the BALF and decreased the levels of IL-6, TNF-α, and ICAM-1 in serum and IL-6, TNF-α, ICAM-1, and P-selectin in BLAF. In addition, baicalin attenuated inflammatory cell infiltration in the lung of ALI mice and reduced the deposition of complement activation products (C5a, C5b-9) and phosphorylation of NF-κB p65 in lung tissue. CONCLUSION: Baicalin relieves complement alternative pathway activation-induced lung inflammation by inhibition of NF-κB pathway, delaying the progression of ALI.

Diterpenoids of Caryopteris trichosphaera W. W. Sm. inhibiting MRSA and VRE in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Caryopteris trichosphaera W. W. Sm., a traditional ethnic medicine, was recorded in the Compendium of Materia Medica for treating wound infection by pathogenic infection. However, its antibacterial potential and bioactive compositions against drug-resistant bacteria need to be validated. AIM OF THE STUDY: To investigate the chemical constituents of C. trichosphaera and explore its anti-MRSA component in vitro and in vivo, together with the antibacterial mechanism. MATERIALS AND METHODS: Bioactive constituents investigation was carried out by phytochemical method and antibacterial screening. The antibacterial mechanism was predicted by network pharmacology, which was further validated by time-kill analysis, membrane function tests, multigenerational resistance induction assay and biofilm test, and metabolomics analysis in vitro. In addition, MRSA-induced epidermal infection in mice was selected to evaluate its pharmacological effect in vivo. RESULTS: Six antibacterial diterpenoids against MRSA and VRE with MIC values 4-32 µg/mL from C. trichosphaera were reported for the first time, in which the major compound cativic acid (1) disrupted MRSA cell membranes by modulating permeability, depolarization, and fluidity while increasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels. It also displayed remarkable anti-biofilm activity without inducing bacterial resistance or cytotoxicity. Moreover, cativic acid affected MRSA biosynthesis of cofactors, amino acid biosynthesis, nucleotide metabolism by metabolomics analysis. Furthermore, cativic acid accelerated wound healing in MRSA-infected mouse skin wounds, even better than vancomycin. CONCLUSIONS: The results supported the traditional use of C. trichosphaera, and presented unreported anti-MRSA agent, cativic acid, as a plant-derived bactericide in vitro and in vivo for the first time.

6-Methoxyldihydrochelerythrine Chloride Inhibiting Intra and Extracellular Drug-Resistant Bacteria.

Vancomycin-resistant enterococcus (VRE) is a major nosocomial pathogen that exhibits enhanced infectivity due to its robust virulence and biofilm-forming capabilities. In this study, 6-methoxyldihydrochelerythrine chloride (6-MDC) inhibited the growth of exponential-phase VRE and restored VRE's sensitivity to vancomycin. 6-MDC predominantly suppressed the de novo biosynthetic pathway of pyrimidine and purine in VRE by the RNA-Seq analysis, resulting in obstructed DNA synthesis, which subsequently weakened bacterial virulence and impeded intracellular survival. Furthermore, 6-MDC inhibited biofilm formation, eradicated established biofilms, reduced virulence, and enhanced the host immune response to prevent intracellular survival and replication of VRE. Finally, 6-MDC reduced the VRE load in peritoneal fluid and cells significantly in a murine peritoneal infection model. This paper provides insight into the potential antimicrobial target of benzophenanthridine alkaloids for the first time.

Thrombosis inhibited by Corydalis decumbens through regulating PI3K-Akt pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis decumbens (Thunb.) Pers. was used as stasis-eliminating medicine traditionally to treat cardiovascular disease potentially attributed to its antithrombotic effect, but lack of pharmacological research on it. AIM OF THE STUDY: To investigate the antithrombotic effect of C. decumbens and its preliminary mechanism. MATERIALS AND METHODS: A carrageenan-induced mouse thrombus model and adenosine diphosphate stimulated platelet aggregation of rabbits were used to confirm the inhibitory effect of C. decumbens extract and compounds on thrombosis in vivo. Then, H2O2-induced human umbilical vein endothelial cells (HUVECs) injury model was further adopted to verify the effects of bioactive compounds in vitro. Moreover, in silico network pharmacology analyses and molecular docking were performed to predict the underlying mechanisms, targets, and pathways, and which were further confirmed through western blotting assay. RESULTS: The administration of total extract (TE), total alkaloids (TA) and tetrahydropalmatine (TET) resulted in a significant reduction in black tail thrombus and congestion, along with a decreasing in platelet aggregation of rabbits. A superior antithrombotic effect indicated the bioactive fraction, and then the isolated bioactive compounds, TET and protopine (PRO) increased cell survival, and decreased reactive oxygen species (ROS) and lactate dehydrogenase (LDH) release in H2O2-induced HUVECs injury model. Moreover, the two alkaloids targeted 33 major proteins and influenced 153 pathways in network pharmacology prediction. Among these, HSP90AA1, COX-2, NF-κB/p65, MMP1 and HIF-1α were the key proteins and PI3K-Akt emerged as the major signaling pathway. Further western blotting results supported that five key proteins were downregulated by the two bioactive compounds in H2O2-stimulated HUVECs model. CONCLUSION: C. decumbens exerted protective effect on thrombosis through inhibiting PI3K-Akt pathway and related key proteins, which supported the traditional use and presented potential antithrombotic alkaloids for further investigation.

The whitening effect of cuscutin responsible for traditional use of Bergenia purpurascens.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and rhizomes of Bergenia purpurascens (Hook. f. et Thomson) Engl., was used as a sunscreen to protect against ultraviolet rays in Tibet of China historically, but its skin whitening constituents and pharmacological effects of this plant remained unknown. AIM OF THE STUDY: To investigate the anti-melanogenesis effect of B. purpurascens in vitro and in vivo, and then explore the preliminary mechanism. MATERIALS AND METHODS: An ultraviolet B (UVB)-induced skin injury model of mice was used to verify the ameliorative effect of B. purpurascens extract (BPE) on ultraviolet damage. Then, alpha-melanocyte stimulating hormone (α-MSH)-induced murine melanoma cell line (B16F10) melanin generation model was further adopted to approval the effects of BPE and its bioactive compound, cuscutin, in vitro. Moreover, α-MSH stimulated melanogenesis model in zebrafish was employed to confirm the anti-pigmentation effect of cuscutin. Then, proteins expressions associated with melanin production were observed using western blotting assay to explore preliminary mechanism. RESULTS: BPE inhibited UVB-induced mice injury and restored skin barrier function observably in vivo. BPE and cuscutin suppressed the overproduction of melanin in α-MSH induced B16F10 significantly, in which cuscutin exhibited better effect than well-known whitening agent α-arbutin at same 10 µg/mL concentration. Moreover, the pigmentation of zebrafish embryo was decreased by cuscutin. Finally, cuscutin showed significant downregulation of expressions of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1), TRP-2 and microphthalmia-associated transcription factor (MITF) in the melanogenic signaling pathway. CONCLUSION: B. purpurascens extract and its major bioactive constituent, cuscutin, showed potent anti-melanogenesis and skin-whitening effect by targeting TYR and TRP-2 proteins for the first time, which supported its traditional use.

New Monoterpenoid Indole Hybrids from Gelsemium elegans with Anti-Inflammatory and Osteoclast Inhibitory Activities.

Gelsegansymines A (1) and B (2), two new indole alkaloids along with six known analogues (3-8) were isolated from the aerial parts of Gelsemium elegans. Their structures were elucidated by means of spectroscopic techniques. Structurally, compounds 1 and 2 possessed the rare cage-like gelsedine skeleton hybrid with bicyclic monoterpenoid. The anti-inflammatory activities of isolated compounds (1-3) were tested on LPS induced RAW264.7 cells. Under the treated concentration without toxicity for cells, the cytokines levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were evaluated by Griess method and enzyme-linked immunosorbent assay (ELISA). The results showed that compounds 1-3 exhibited anti-inflammatory activities with dose-dependent manner range from 12.5 to 50 µmol/L. Furthermore, the inhibitory activities of compounds 1 and 2 on receptor activator of NF-κB ligand (RANKL) induced osteoclast formation were tested in vitro. Compounds 1 and 2 at 5 µmol/L exhibited the significant inhibitory effect on the osteoclastogenesis induced by RANKL. This work reported the anti-inflammatory and osteoclast inhibitory activities of new monoterpenoid indole hybrids, which may inspire the further light on the related traditional application research of G. elegans.

Cobra venom P-III class metalloproteinase atrase A induces inflammatory response and cell apoptosis in endothelial cells via its metalloproteinase domain.

Snake venom metalloproteinases (SVMPs), which are a critical component of viperid and crotalid venoms, play various important roles in the pathogenesis of snakebite envenomation. The SVMPs from elapid venoms are not well elucidated, as compared with those from viperid and crotalid venoms. Atrase A is a nonhemorrhagic P-III SVMP purified from Naja atra venom that possesses only weak fibrinogenolytic activity. In our prior study, we found that atrase A detached adherent cells from the substrate. In this work, we investigated further the effect and mechanism of atrase A on endothelial cells. Oxidative damage, inflammatory mediators, apoptosis, and activation of the NF-κB and MAPK signaling pathways were measured after HMEC-1 cells were exposed to atrase A. The results showed that HMEC-1 cells released inflammatory mediators, exihibited oxidative damage and apoptosis after exposure to atrase A. The Western blot analysis results revealed that atrase A increased Bax/Bcl-2 and caspase-3 levels and activated the NF-κB and MAPK signaling pathways in endothelial cells. The effects on endothelial cells were nearly completely abolished after atrase A was treated with ethylenediamine tetraacetic acid. These results showed that atrase A led to an inflammatory response, cellular injury and apoptosis in endothelial cells, and this effect was due to its metalloproteinase domain. The study contributes to a better understanding of the structures and functions of cobra venom P-III class metalloproteinases.

Salvianolic acid A alleviates lipopolysaccharide-induced disseminated intravascular coagulation by inhibiting complement activation.

INTRODUCTION: Disseminated intravascular coagulation (DIC) is a syndrome characterized by coagulopathy, microthrombus, and multiple organ failure. The complement system in DIC is overactivated, and the functions of complement and coagulation pathways are closely related. Our previous screening revealed that salvianolic acid A (SAA) has anti-complement activity. The hyper-activated complement system was involved in the lipopolysaccharide (LPS) induced DIC in rats. The effects of SAA anti-complement action on LPS-induced DIC in rats were investigated. METHODS: The complement activity of the classical pathway and alternative pathway was detected through an in vitro hemolysis assay. The binding sites of SAA and complement C3b were predicted by molecular docking. LPS-induced disseminated coagulation experiments were performed on male Wistar rats to assess coagulation function, complement activity, inflammation, biochemistry, blood routine, fibrinolysis, and survival. RESULTS: SAA had an anti-complement activity in vivo and in vitro and inhibited the complement activation in the classical and alternative pathway of complement. The infusion of LPS into the rats impaired the coagulation function, increased the plasma inflammatory cytokine level, complemented activation, reduced the clotting factor levels, fibrinogen, and platelets, damaged renal, liver, and lung functions, and led to a high mortality rate (85%). SAA treatment of rats inhibited complement activation and attenuated the significant increase in D-dimer, interleukin-6, alanine aminotransferase, and creatinine. It ameliorated the decrease in plasma levels of fibrinogen and platelets and reversed the decline in activity of protein C and antithrombin III. The treatment reduced kidney, liver, and lung damage, and significantly improved the survival rate of rats (46.2 and 78.6% for the low- and high-dose groups, respectively). CONCLUSION: SAA reduced LPS-induced DIC by inhibiting complement activation. It has considerable potential in DIC treatment.