Huan-Huan Zhang1, Wen-Ying Yu1, Lan Li2, Fang Wu3, Qin Chen4, Yang Yang1, Chen-Huan Yu5. 1. Key Laboratory of Experimental Animal and Safety Evaluation, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China. 2. First Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou 310053, China. 3. Key Laboratory of Experimental Animal and Safety Evaluation, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China; First Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou 310053, China. 4. Department of Clinical Laboratory Medicine, Second Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310009, China. 5. Key Laboratory of Experimental Animal and Safety Evaluation, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China. Electronic address: yuchenhuan2002@163.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Moslae Herba (MH) is broadly used as an antiviral, antipyretic and anticoagulant drug which effectively treats respiratory diseases including cough, asthma, throat, cold and flu. AIM OF THIS STUDY: The excessive inflammation of the lungs is the hallmark of severe influenza A virus (IAV) infection, while platelet aggregation and its subsequent microvascular thrombosis can exacerbate IAV-induced lung injury. Thus, inhibition of platelet aggregation can be a potential target for IAV treatment. Previous studies focus on the flavonoids from MH and their anti-inflammatory activities, but the anticoagulant compounds and potential molecular mechanism of MH remains unclear. This study was to isolate and characterize diketopiperazines (DKPs) from MH and to explore the underlying anticoagulant mechanism on IAV infection models. MATERIALS AND METHODS: EtOAc sub-extract separated from MH ethanolic extract was subjected to fractionation through column chromatography. The chemical structures of pure compounds were characterized by the spectral analysis. Antiviral activities of DKPs were assayed in IAV-infected Madin-Darby canine kidney (MDCK) cells and mice. Anticoagulant effects of DKPs were investigated on adenosine 5'-diphosphate (ADP)-induced acute pulmonary embolism and IAV-induced lung injury in vivo, as well as the inhibition on platelet activating factor (PAF), arachidonic acid (AA) and ADP-induced platelet aggregation in vitro. The serum levels of thromboxane B2 (TXB2) and 6-keto-PGF1α were detected by ELISA. The expressions of key proteins in CD41-mediated PI3K/AKT pathways were determined by western blotting analysis. RESULTS: Six DKPs were, for the first time, isolated from MH and identified as cyclo(Tyr-Leu) (1), cyclo(Phe-Phe) (2), cyclo(Phe-Tyr) (3), cyclo(Ala-Ile) (4), cyclo(Ala-Leu) (5) and Bz-Phe-Phe-OMe (6). Among these DKPs, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe possessed low cytotoxicities and significant inhibition against cytopathic effects induced by IAV (H1N1 and H3N2) replication in MDCK cells. Furthermore, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe significantly alleviated IAV-induced platelet activation and lung inflammation in mice. They could reduce the expression of CD41 and the phosphorylation of PI3K and AKT in PLTs of IAV-infected mice. CONCLUSION: These results suggested that cyclo(Ala-Ile) and Bz-Phe-Phe-OMe isolated from MH have antiviral and anticoagulant effects against IAV-induced PLT aggregation and lung inflammation via regulating CD41/PI3K/AKT pathway, and could be used as the potential agents for IAV treatment.
ETHNOPHARMACOLOGICAL RELEVANCE: Moslae Herba (MH) is broadly used as an antiviral, antipyretic and anticoagulant drug which effectively treats respiratory diseases including cough, asthma, throat, cold and flu. AIM OF THIS STUDY: The excessive inflammation of the lungs is the hallmark of severe influenza A virus (IAV) infection, while platelet aggregation and its subsequent microvascular thrombosis can exacerbate IAV-induced lung injury. Thus, inhibition of platelet aggregation can be a potential target for IAV treatment. Previous studies focus on the flavonoids from MH and their anti-inflammatory activities, but the anticoagulant compounds and potential molecular mechanism of MH remains unclear. This study was to isolate and characterize diketopiperazines (DKPs) from MH and to explore the underlying anticoagulant mechanism on IAV infection models. MATERIALS AND METHODS:EtOAc sub-extract separated from MH ethanolic extract was subjected to fractionation through column chromatography. The chemical structures of pure compounds were characterized by the spectral analysis. Antiviral activities of DKPs were assayed in IAV-infected Madin-Darby canine kidney (MDCK) cells and mice. Anticoagulant effects of DKPs were investigated on adenosine 5'-diphosphate (ADP)-induced acute pulmonary embolism and IAV-induced lung injury in vivo, as well as the inhibition on platelet activating factor (PAF), arachidonic acid (AA) and ADP-induced platelet aggregation in vitro. The serum levels of thromboxane B2 (TXB2) and 6-keto-PGF1α were detected by ELISA. The expressions of key proteins in CD41-mediated PI3K/AKT pathways were determined by western blotting analysis. RESULTS: Six DKPs were, for the first time, isolated from MH and identified as cyclo(Tyr-Leu) (1), cyclo(Phe-Phe) (2), cyclo(Phe-Tyr) (3), cyclo(Ala-Ile) (4), cyclo(Ala-Leu) (5) and Bz-Phe-Phe-OMe (6). Among these DKPs, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe possessed low cytotoxicities and significant inhibition against cytopathic effects induced by IAV (H1N1 and H3N2) replication in MDCK cells. Furthermore, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe significantly alleviated IAV-induced platelet activation and lung inflammation in mice. They could reduce the expression of CD41 and the phosphorylation of PI3K and AKT in PLTs of IAV-infectedmice. CONCLUSION: These results suggested that cyclo(Ala-Ile) and Bz-Phe-Phe-OMe isolated from MH have antiviral and anticoagulant effects against IAV-induced PLT aggregation and lung inflammation via regulating CD41/PI3K/AKT pathway, and could be used as the potential agents for IAV treatment.
Authors: Joanna Bojarska; Adam Mieczkowski; Zyta M Ziora; Mariusz Skwarczynski; Istvan Toth; Ahmed O Shalash; Keykavous Parang; Shaima A El-Mowafi; Eman H M Mohammed; Sherif Elnagdy; Maha AlKhazindar; Wojciech M Wolf Journal: Biomolecules Date: 2021-10-14