Design, synthesis, and activity evaluation of novel multitargeted l-tryptophan derivatives with powerful antioxidant activity against Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial neurological disease, and the multitarget directed ligand (MTDL) strategy may be an effective approach to delay its progression. Based on this strategy, 27 derivatives of l-tryptophan, 3a-1-3d-1, were designed, synthesized, and evaluated for their biological activity. Among them, IC50 (inhibitor concentration resulting in 50% inhibitory activity) values of compounds 3a-18 and 3b-1 were 0.58 and 0.44 µM for human serum butyrylcholinesterase (hBuChE), respectively, and both of them exhibited more than 30-fold selectivity for human serum acetylcholinesterase. Enzyme kinetics studies showed that these two compounds were mixed inhibitors of hBuChE. In addition, these two derivatives possessed extraordinary antioxidant activity in OH radical scavenging and oxygen radical absorption capacity fluorescein assays. Meanwhile, these compounds could also prevent ß-amyloid (Aß) self-aggregation and possessed low toxicity on PC12 and AML12 cells. Molecular modeling studies revealed that these two compounds could interact with the choline binding site, acetyl binding site, and peripheral anionic site to exert submicromolar BuChE inhibitory activity. In the vitro blood-brain barrier permeation assay, compounds 3a-18 and 3b-1 showed enough blood-brain barrier permeability. In drug-likeness prediction, compounds 3a-18 and 3b-1 showed good gastrointestinal absorption and a low risk of human ether-a-go-go-related gene toxicity. Therefore, compounds 3a-18 and 3b-1 are potential multitarget anti-AD lead compounds, which could work as powerful antioxidants with submicromolar selective inhibitory activity for hBuChE as well as prevent Aß self-aggregation.

Carpelipines C and D, Two Anti-Inflammatory Germacranolides from the Flowers of Carpesium lipskyi Winkl. (Asteraceae).

Two new germacranolides, carpelipine C (1) and carpelipine D (2), together with four known ones (3-6), were isolated from Carpesium lipskyi Winkl. flowers, a folk Tibetan herbal medicine with antipyretic-analgesic and anti-inflammatory effects. The chemical structures of new structure were illuminated by diversified spectroscopic and X-ray crystallographic analyses. Compounds 1 and 3 dramatically suppressed the synthesis of NO and decreased pre-inflammatory protein expression of iNOS and COX-2 in LPS-induced RAW264.7 cells. Furthermore, it was revealed that NF-κB/MAPK signaling pathway were involved in the anti-inflammatory process of 1 and 3, and their effects on reducing oxidative stress by activating Nrf2/HO-1 pathway were also measured. This article indicated that the traditional use of C. lipskyi to treat inflammatory diseases has a certain rationality.

Diethyl Blechnic Exhibits Anti-Inflammatory and Antioxidative Activity via the TLR4/MyD88 Signaling Pathway in LPS-Stimulated RAW264.7 Cells.

Inflammation is a common pathogenesis in many diseases. Salvia miltiorrhiza Bunge (Danshen), a traditional Chinese medicine, has been considered to have good anti-inflammatory effects. In the present study, we investigated the anti-inflammatory effect of diethyl blechnic (DB), a novel compound isolated from Danshen, and its possible mechanisms in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. The results showed that DB can inhibit the LPS-induced pro-inflammatory cytokines release of prostaglandin E2 (PGE2) and mRNA expression of TNF-α, IL-6, and IL-1ß. In addition, the results of the flow cytometry assay and the fluorometric intracellular ROS kit assay indicated that DB reduced the generation of ROS in LPS-stimualted RAW264.7 cells. DB reversed the LPS-induced loss of the mitochondrial membrane potential (MMP). Furthermore, DB suppressed the LPS-stimulated increased expression of Toll-like receptor 4 (TLR4), myeloid differential protein-88 (MyD88) and phosphorylation of TAK1, PI3K, and AKT. DB promoted NF-E2-related factor 2 (Nrf2) into the nucleus, increased the expression of heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) and reduced the expression of Keap1. In summary, DB may inhibit LPS-induced inflammation, which mainly occurs through TLR4/MyD88 and oxidative stress signaling pathways in RAW264.7 cells.

Characterization of chemical constituents and metabolites in rat plasma after oral administration of Ainsliaea fragrans Champ by using UHPLC-QTOF-MS/MS.

Ainsliaea fragrans Champ, a strong heat-clearing and detoxifying traditional Chinese medicine, has been effectively used for treating chronic cervicitis, endometritis, pelvic inflammatory diseases, and other conditions caused by damp heat. It shows a good effect in the treatment of cervicitis and has broad clinical application prospects. Nevertheless, there is no comprehensive study on its in vivo and in vitro chemical analysis. UHPLC-QTOF-MS/MS combined with the non-targeted characteristic filter analysis were used to conjecture and characterize the chemical components and in vivo metabolites of rats following oral administration of Ainsliaea fragrans Champ. In this study, A total of 85 compounds were identified in Ainsliaea fragrans Champ, including 29 flavonoids, 14 sesquiterpenoids, 25 chlorogenic acids, and 17 other compounds. In the plasma of rats after administration of Ainsliaea fragrans Champ, 160 compounds were deduced (19 prototype compounds and 141 metabolites). The 141 metabolites consist of 50 flavonoids, 80 phenolic acids and 11 Chlorogenic acids. The related metabolic pathways mainly involved demethylation, reduction, sulfonation, decarboxylation, hydroxylation, methylation, and glucuronide conjunction. In summary, the chemical components and metabolites of Ainsliaea fragrans Champ were comprehensively identified by using a rapid and accurate analysis method, which laid a foundation for dissecting its bioactive substances. In addition, it provides a scientific basis for the in-depth study of the material basis of Ainsliaea fragrans Champ efficacy and theoretical support for illustrating the mechanism of medical action and its clinical application.

San Pian decoction can treat nitroglycerin-induced migraine in rats by inhibiting the PI3K/AKT and MAPK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: San Pian decoction (SPD), a traditional Chinese medicine preparation composed of eight herbs, has been reported to alleviate migraine. However, its active ingredients and the potential mechanism of action remains unclear. The purpose of this study was to comprehensively analyze SPD for the treatment of chronic migraine based on pharmacological direction and to identify the active ingredients and pharmacological mechanism of SPD in the treatment of migraine. MATERIALS AND METHODS: The active components in SPD were identified by AB SCIEX quadrupole time-of-flight mass spectrometer, and the prediction targets and pharmacological networks related to migraine were constructed. The mechanism of SPD in treating migraine was studied through network pharmacology, which was further verified using pharmacological experiments. RESULTS: A total of 489 targets of 26 compounds were identified. Based on Venn analysis, we found 117 intersection targets between SPD and migraine, that is, these targets were related to the treatment of migraine. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the treatment of migraine using SPD was related to the PI3K/AKT and MAPK signaling pathways. The effect of SPD on migraine was verified by measuring the levels of the inflammatory factors, nitric oxide (NO), interleukin (IL-6), endothelin (ET),5-hydroxytryptamine(5-HT), indoleamine 2,3-dioxygenas (IDO), tumor necrosis factor (TNF-α) and calcitonin gene-related peptide (CGRP). Lastly, real-time polymerase chain reaction and western blotting were used to verify gene and protein expression in the PI3K/AKT and MAPK signaling pathways. Expression of the genes P38, JNK, ERK, PI3K and AKT, and the protein expression of p-P38, p-JNK, p-ERK, p-AKT and p-PI3K were significantly downregulated. Our findings indicated that SPD could prevent inflammation by regulating the inflammatory cytokines and key genes and proteins in the PI3K/AKT and MAPK signaling pathways to treat migraine. CONCLUSION: Our findings reveal that SPD could treat nitroglycerin-induced migraine by regulating p-AKT, p-pI3k, p-p38, p-ERK, p-JNK, IL-6, and TNF-α inflammatory factors in the PI3K/AKT and MAPK signaling pathways.

Luteolin-7-O-rutinoside from Pteris cretica L. var. nervosa attenuates LPS/D-gal-induced acute liver injury by inhibiting PI3K/AKT/AMPK/NF-κB signaling pathway.

Pteris cretica L. var. nervosa is one of the most well-known Chinese medicines. Although it is widely used to treat jaundice hepatitis, the main ingredient for its treatment was not thoroughly explored until recently. Essentially, the purpose of this study is to find the monomer compound in Pteris cretica L. var. nervosa, which is most likely to be effective in treating liver injury. Through the model of LPS/D-gal-induced liver injury in mice, the best therapeutic site of the total extract was explored, the chemical components of the parts with the best therapeutic effect were separated, a total of 10 flavonoids were isolated, and the RAW264.7 cells induced by LPS were used as the experimental model to explore the preliminary anti-inflammatory activity of NO production in vitro. Finally, the anti-inflammatory activity and the highest content in this plant Luteolin-7-O-rutinoside (LUT) were selected, as the object of study in vivo. It was found that LUT could not only reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, but also significantly reduce the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), and inhibit PI3K/AKT/AMPK/NF-κB pathway. In addition, LUT can increase levels of SOD and GSH to reduce oxidative stress. It has an obvious therapeutic effect on acute liver injury induced by LPS/D-gal in mice. Therefore, infer LUT is a functional substance in Pteris cretica L. var. nervosa.

Cryptotanshinone Induces Necroptosis Through Ca2+ Release and ROS Production in vitro and in vivo.

BACKGROUND: Necroptosis is a type of programmed necrosis mediated by receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3), which is morphologically characterized by enlarged organelles, ruptured plasma membrane, and subsequent loss of intracellular contents. Cryptotanshinone (CPT), a diterpene quinone compound extracted from the root of Salvia miltiorrhiza Bunge, has been reported to have significant anticancer activities. However, the detailed mechanism of CPT has not been clearly illustrated. OBJECTIVE: The present study aimed to explore the cell death type and mechanisms of CPT-induced in non-small cell lung cancer (NSCLC) cells. METHODS: The cytotoxicity of CPT on A549 cells was assessed by MTS assay. Ca2+ release and reactive oxygen species (ROS) generation were detected by flow cytometry. The changes in mitochondrial membrane potential (MMP) were observed through JC-1 staining. The expressions of p- RIP1, p-RIP3, p-MLKL, and MAPKs pathway proteins were analyzed by western blotting analysis. The efficacy of CPT in vivo was evaluated by the Lewis lung carcinoma (LLC) xenograft mice model. Blood samples were collected for hematology analysis. ELISA investigated the effects of CPT on tumor necrosis factor α (TNF-α). Hematoxylin and eosin staining (HE) determined the tumor tissues. Proteins' expression of tumor tissues was quantified by western blotting. RESULTS: CPT inhibited the cell viability of A549 cells in a time- and concentration-dependent manner, which was reversed by Necrostatin-1 (Nec-1). In addition, CPT treatment increased the expression of p-RIP1, p-RIP3, p-MLKL, the release of Ca2+, ROS generation, and the MAPKs pathway activated in A549 cells. Moreover, animal experiment results showed that intraperitoneal injection of CPT (15 mg/kg and 30 mg/kg) significantly inhibited tumor growth in C57BL/6 mice without affecting the bodyweight and injuring the organs. CONCLUSION: Our findings suggested that CPT-induced necroptosis via RIP1/RIP3/MLKL signaling pathway both in vitro and in vivo, indicating that CPT may be a promising agent in the treatment of NSCLC.

Hederasaponin C Alleviates Lipopolysaccharide-Induced Acute Lung Injury In Vivo and In Vitro Through the PIP2/NF-κB/NLRP3 Signaling Pathway.

Gene transcription is governed by epigenetic regulation that is essential for the pro-inflammatory mediators surge following pathological triggers. Acute lung injury (ALI) is driven by pro-inflammatory cytokines produced by the innate immune system, which involves the nod-like receptor 3 (NLRP3) inflammasome and nuclear factor-κB (NF-κB) pathways. These two pathways are interconnected and share a common inducer the phosphatidylinositol 4,5-bisphosphate (PIP2), an epigenetic regulator of (Ribosomal ribonucleic acid (rRNA) gene transcription, to regulate inflammation by the direct inhibition of NF-κB phosphorylation and NLRP3 inflammasome activation. Herein, we report that hederasaponin C (HSC) exerted a therapeutic effect against ALI through the regulation of the PIP2/NF-κB/NLRP3 signaling pathway. In lipopolysaccharide (LPS)/lipopolysaccharide + adenosine triphosphate (LPS+ATP)-stimulated macrophages, our results showed that HSC remarkably inhibited the secretion of interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α). Moreover, HSC inhibited NF-κB/p65 nuclear translocation and the binding of PIP2 to transforming growth factor-ß activated kinase 1 (TAK1). The intracellular calcium (Ca2+) level was decreased by HSC via the PIP2 signaling pathway, which subsequently inhibited the activation of NLRP3 inflammasome. HSC markedly alleviated LPS-induced ALI, restored lung function of mice, and rescued ALI-induced mice death. In addition, HSC significantly reduced the level of white blood cells (WBC), neutrophils, and lymphocytes, as well as pro-inflammatory mediators like IL-6, IL-1ß, and TNF-α. Hematoxylin and eosin (H&E) staining results suggested HSC has a significant therapeutic effect on lung injury of mice. Interestingly, the PIP2/NF-κB/NLRP3 signaling pathway was further confirmed by the treatment of HSC with ALI, which is consistent with the treatment of HSC with LPS/LPS+ATP-stimulated macrophages. Overall, our findings revealed that HSC demonstrated significant anti-inflammatory activity through modulating the PIP2/NF-κB/NLRP3 axis in vitro and in vivo, suggesting that HSC is a potential therapeutic agent for the clinical treatment of ALI.

Oleuropein Attenuates Lipopolysaccharide-Induced Acute Kidney Injury In Vitro and In Vivo by Regulating Toll-Like Receptor 4 Dimerization.

Acute kidney injury (AKI) is a common critical illness that involves multiple systems and multiple organs with a rapid decline in kidney function over short period. It has a high mortality rate and presents a great treatment challenge for physicians. Oleuropein, the main active constituent of Ilex pubescens Hook. et Arn. var. kwangsiensis Hand.-Mazz. displays significant anti-inflammatory activity, although oleuropein's therapeutic effect and mechanism of action in AKI remain to be elucidated. The present study aimed to further clarify the mechanism by which oleuropein exerts effects on inflammation in vitro and in vivo. In vitro, the inflammatory effect and mechanism were investigated through ELISA, Western blotting, the thermal shift assay, co-immunoprecipitation, and immunofluorescence staining. Lipopolysaccharide (LPS) induced acute kidney injury was employed in an animal model to investigate oleuropein's therapeutic effect on AKI and mechanism in vivo. The underlying mechanisms were investigated by Western blot analysis of kidney tissue. In LPS-stimulated macrophages, our data demonstrated that oleuropein significantly reduced the expression of inflammatory mediators like NO, IL-6, TNF-α, iNOS, and COX-2. Moreover, oleuropein inhibited NF-κB/p65 translocation, and had a negative regulatory effect on key proteins in the NF-κB and MAPK pathways. In addition, the thermal shift and co-immunoprecipitation assays revealed that oleuropein played an essential role in binding to the active sites of TLR4, as well as inhibiting TLR4 dimerization and suppressing the binding of TLR4 to MyD88. Oleuropein markedly alleviated LPS induced acute kidney injury, decreased serum creatinine and blood urea nitrogen (BUN) levels and proinflammatory cytokines. More importantly, the TLR4-MyD88-NF-κB/MAPK pathways were confirmed to play an important role in the oleuropein treatment of AKI. In this study, oleuropein exhibited excellent anti-inflammatory effects by regulating TLR4-MyD88-NF-κB/MAPK axis in vitro and in vivo, suggesting oleuropein as a candidate molecule for treating AKI.

Anemoside B4 protects against Klebsiella pneumoniae- and influenza virus FM1-induced pneumonia via the TLR4/Myd88 signaling pathway in mice.

BACKGROUND: Pneumonia refers to the inflammation of the terminal airway, alveoli and pulmonary interstitium, which can be caused by pathogenic microorganisms, physical and chemical factors, immune damage, and drugs. Anemoside B4, the major ingredient of Pulsatilla chinensis (Bunge) Regel, exhibited anti-inflammatory activity. However, the therapeutic effect of anemoside B4 on pneumonia has not been unraveled. This study aims to investigate that anemoside B4 attenuates the inflammatory responses in Klebsiella pneumonia (KP)- and influenza virus FM1 (FM1)-induced pneumonia mice model. METHODS: The network pharmacology and molecular docking assays were employed to predict the targets of anemoside B4's treatment of pneumonia. Two models (bacterial KP-infected mice and virus FM1-infected mice) were employed in our study. BALB/c mice were divided into six groups: control, model group (KP-induced pneumonia or FM1-induced pneumonia), anemoside B4 (B4)-treated group (2.5, 5, 10 mg/kg), and positive drug group (ribavirin or ceftriaxone sodium injection). Blood samples were collected for hematology analysis. The effects of B4 on inflammation-associated mediators were investigated by Enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin staining (HE) staining. Proteins expression was quantified by western blotting. RESULTS: The network results indicated that many pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) participated in anemoside B4's anti-inflammatory activity. The counts of neutrophil (NEU) and white blood cell (WBC), the level of myeloperoxidase (MPO), and the release of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 increased by KP or FM1 infection, which were reversed by anemoside B4. In addition, anemoside B4 significantly suppressed the FM1-induced expression of toll-like receptor 4 (TLR4), myeloid differential protein-88 (MyD88), and myeloid differentiation protein-2 (MD-2), which were further validated by molecular docking data that anemoside B4 bound to bioactive sites of TLR4. Therefore, anemoside B4 exhibited a significant therapeutic effect on pneumonia via the TLR4/MyD88 pathway. CONCLUSION: Our findings demonstrated that anemoside B4 attenuates pneumonia via the TLR4/Myd88 signaling pathway, suggesting that anemoside B4 is a promising therapeutic candidate for bacterial-infected or viral-infected pneumonia.