Atish Gheware, Dhwani Dholakia, Sadasivam Kannan, Lipsa Panda, Ritu Rani, Bijay Ranjan Pattnaik, Vaibhav Jain, Yash Parekh, M. Ghalib Enayathullah, Kiran Kumar Bokara, Venkatesan Subramanian, Mitali Mukerji, Anurag Agrawal & Bhavana Prasher
Abstract
Background
COVID-19 pneumonia has been associated with severe acute hypoxia, sepsis-like states, thrombosis and chronic sequelae including persisting hypoxia and fibrosis. The molecular hypoxia response pathway has been associated with such pathologies and our recent observations on anti-hypoxic and anti-inflammatory effects of whole aqueous extract of Adhatoda Vasica (AV) prompted us to explore its effects on relevant preclinical mouse models.
Methods
In this study, we tested the effect of whole aqueous extract of AV, in murine models of bleomycin induced pulmonary fibrosis, Cecum Ligation and Puncture (CLP) induced sepsis, and siRNA induced hypoxia-thrombosis phenotype. The effect on lung of AV treated naïve mice was also studied at transcriptome level. We also determined if the extract may have any effect on SARS-CoV2 replication.
Results
Oral administration AV extract attenuates increased airway inflammation, levels of transforming growth factor-β1 (TGF-β1), IL-6, HIF-1α and improves the overall survival rates of mice in the models of pulmonary fibrosis and sepsis and rescues the siRNA induced inflammation and associated blood coagulation phenotypes in mice. We observed downregulation of hypoxia, inflammation, TGF-β1, and angiogenesis genes and upregulation of adaptive immunity-related genes in the lung transcriptome. AV treatment also reduced the viral load in Vero cells infected with SARS-CoV2.
Conclusion
Our results provide a scientific rationale for this ayurvedic herbal medicine in ameliorating the hypoxia-hyperinflammation features and highlights the repurposing potential of AV in COVID-19-like conditions.
Background
Increased alveolar hypoxic response levels are inevitable consequences of many respiratory disorders such as chronic obstructive pulmonary disease and pulmonary fibrosis. The key player of cellular response to hypoxia is the hypoxia-inducible factor (HIF)-1α and its regulatory protein, the prolyl hydroxylase domain (PHD)-2 enzyme. The induction of HIF-1α is considered to be pro-inflammatory. It leads to transcriptional activation of essential genes implicated in airway remodelling and inflammation, such as vascular endothelial growth factor, transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase, interleukin -17 (IL-17), and IL-6 . Thus, it is not just a consequence of diseases, elevated tissue/cellular hypoxia actively participates in exaggerating the inflammatory response contributing to progressive lung damage/injury.
HIF-1α also plays a pivotal role in infection, especially in promoting viral and bacterial replication. In the present COVID-19 pandemic caused by the severe acute respiratory coronavirus 2 (SARS-CoV2), the role of hypoxia response in inducing severe lung inflammation and other outcomes has been one of the most highlighted observations. Clinically, the interaction of the host and SARS-CoV2 is broadly described in three stages: first, asymptomatic state; second, a non-severe symptomatic state characterized by upper airway and conducting airway response; third, severe respiratory symptomatic state with the presence of hypoxia, acute respiratory distress syndrome (ARDS) and progression to sepsis . During incubation and non-severe state, a specific humoral and cell-mediated adaptive immune response is required to eradicate the virus and prevent disease progression to a severe condition. Thus, strategies to boost immune responses at this stage are undoubtedly important . However, defective immune response causes further accumulation of immune cells in the lungs, progressing to aggressive production of a pro-inflammatory cytokine such as IL-6, TNF-α resulting in an influx of immune cells and cytokines that damage the airways/ lung architecture. This extended release of cytokines by the immune system in response to the viral infection and/or secondary infections causes severe inflammation, endothelial dysfunction, sepsis and multi-organ damage . In addition, recent research also reports coagulation abnormalities in severe COVID-19 cases . The relation of hypoxia-coagulation is well known, where we and others also showed the crucial role of cellular hypoxic response in the form of thrombosis and bleeding susceptibility through HIF-1α and vWF axis . Thus, medicinal agents that possess immune-boosting and anti-hypoxic effects could hold a promise for a better therapeutic option to preclude the SARS-CoV2 infection and severity.
We have recently shown an extract of Adhatoda Vasica (AV); an ayurvedic medicine that possesses robust anti-hypoxic properties and can reduce severe airway inflammation induced by an augmented hypoxic response in treatment-resistant asthmatic mice . The anti-HIF-1α effect of AV also restores the cellular hypoxia-mediated loss of mitochondrial morphofunction in vitro. As a follow-up, we evaluated AV’s usefulness in other severe lung pathologies, where hypoxia signalling is pertinent, and which are relevant to the clinical course of COVID-19 namely lung injury, fibrosis, and thrombosis. Since viral proliferation may be altered by molecular modulation of such pathways, we further tested the potential of AV in limiting SARS-CoV2 proliferation.
Methods
Preparation of plant extract and LC–MS fingerprinting
Adhatoda Vasica (AV) was collected from Delhi-NCR region, India in the flowering season (November to March). Water extract of plant (leaves, twigs and flowers) was prepared according to classical method described for rasakriya in Caraka Samhita . The process for the formulation involved preparation of decoction condensation and drying as described in earlier study . Chemical fingerprinting of prepared AV extract was carried out by LC–MS at SAIF, CSIR-CDRI, Lucknow, India.
Animals
The study was designed and performed following guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and approved by Institutional Animal Ethics Committee of CSIR-Institute of Genomics & Integrative Biology (IGIB), New Delhi, India. The BALB/c and C57BL/6 male mice (8–10 weeks old) were bred under the pathogen-free condition. They were acclimatized to animal house environment one week before starting the experiments at CSIR-IGIB, New Delhi, India and maintained according to guidelines of CPCSEA. All the surgical procedures were performed under sodium pentobarbital anaesthesia and maximum efforts are taken for minimum suffering of animals.
Grouping and treatment of mice
Mice were mainly divided into two groups as Vehicle and treatment according to the experiment. In the case of Cecum ligation puncture (CLP) survival study experiment, BALB/c mice were divided into Sham (Control mice, distil water and 10% ethanol, oral, n = 5) and CLP (mice underwent CLP surgery, n = 9). CLP mice subdivide in CLP + Cyclo A (Cyclosporin A treated CLP mice, n = 9) and CLP + AV-D2 (Adhatoda Vasica extract-treated CLP mice, n = 9) group. Treatment of AV (130 mg/kg dissolved in distilled water, oral, two times a day) or Cyclo A (Cyclosporin A, 15 mg/kg dissolved in 10% ethanol, oral, once a day) was started two days (48hours) before CLP and was continued till the mice survive after surgery (Fig. 1). To assess lung histology and cytokine levels in the CLP experiment, mice sacrificed after 20 h of surgery from Sham (n = 3), CLP alone (n = 3), CLP + Cyclo A (n = 4), and CLP + AV-D2 (n = 4) group. Similarly, in the bleomycin fibrosis model (n = 5 mice/group), C57BL/6 mice were divided into Vehicle (i.e., Sham), Bleo (bleomycin treated), and Bleo + AV-D2 (AV 130 mg/kg treated Bleo mice, oral, two times a day). In that, AV treatment was done from day 18 to 21, as shown in the schematic (Fig. 1a). Bleomycin (3.5 U/kg of mice) was given intratracheally to isoflurane-anesthetised C57BL/6 mice on day 0 of the protocol (Fig. 1) to induce fibrotic changes in mice as described previously . For transcriptomic study, BALB/c mice were divided into Vehicle (distil water, oral, two times a day, n = 4) and Adhatoda Vasica (AV) extract group. AV group was further subdivided according to its dose: AV-D2 (Adhatoda Vasica extract 130 mg/kg, dissolved in distilled water, oral, two times a day, n = 5) and AV-D4 (Adhatoda Vasica extract 260 mg/kg, dissolved in distilled water, oral, two times a day, n = 5) as described previously . Distil water or AV (130 mg/kg or 260 mg/kg) treatment was given to mice by oral gavage for four consecutive days, as represented in the Fig. 2a. In PHD2 siRNA-induced hypoxia model (n = 5 mice/group), BALB/c mice were divided into scrambled siRNA (Scrm siRNA), prolyl hydroxylase domain-2 siRNA (PHD2 siRNA), and AV-D4 treated PHD2 siRNA group (PHD2 siRNA + AV-D4) group. AV-D4 dose (260 mg/kg, dissolved in distilled water, oral, two times a day) given for four consecutive days and 90 µg siRNA (Sigma) administered intranasally which dissolved in ultrapure DNase and RNAse free water with in-vivo jetPEI as the transfection reagent (Polyplus Transfection, France) to isoflurane-anesthetised mice on day 1, 3 and 5th of the protocol.
Read more at: https://respiratory-research.biomedcentral.com/articles/10.1186/s12931-021-01698-9