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 [15]. The process for the formulation involved preparation of decoction condensation and drying as described in earlier study [16]. Chemical fingerprinting of prepared AV extract was carried out by LC-MS at CSIR-CDRI, Lucknow, India; in two independent experiment.
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 case of Cecum ligation puncture (CLP) experiment (n = 5-9), BALB/c mice were divided in Vehicle ( distil water and 10% ethanol, oral) and CLP (mice underwent CLP surgery). CLP mice subdivide in CLP+Cyclo A (Cyclosporin A treated CLP mice) and CLP+AV-D2 (Adhatoda extract treated CLP mice) group. For CLP experiment, treatment of AV (130mg/kg dissolved in distilled water, oral) or Cyclo A ( Cyclosporin A, 15mg/kg dissolved in 10% ethanol, orall) was started two days (48hours) before CLP and was continued till the mice survives after CLP (figure 1). In that some mice (n=3-4) from each group were sacrificed after 20 hours of CLP to assess lung histology and cytokine levels. Similarly, in bleomycin fibrosis model (n = 5), C57BL/6 mice were divided in Vehicle (i.e. Sham), Bleo (bleomycin treated) and Bleo+AV-D2 (AV 130mg/kg treated Bleo mice). In that AV treatment was done from day 18 to 21, as shown in the schematic (figure 1A). Bleomycin (3.5 U/kg of mice) was given intratracheally to isoflurane-anesthetised C57BL/6 mice on day 0 of the protocol (figure 1) to induce fibrotic changes in mice as described previously [17]. For transcriptomic research (n = 4-5), BALB/c mice divided into Vehicle (distil water, oral) and Adhatoda Vasica (AV) extract group. AV group is further subdivided into two according to its dose: AV-D2 (Adhatoda Vasica extract 130 mg/kg, dissolved in distilled water, oral) and AV-D4 (Adhatoda Vasica extract 260 mg/kg, dissolved in distilled water, oral) as described previously [14]. Distil water or AV ( 130mg/kg or 260mg/kg) treatment was given to mice by oral gavage for consecutive four days as represented in the figure. In PHD2 siRNA induced hypoxia model (n = 4-5) BALB/c mice were divided in 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) given for four consecutive days and 90µg 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.
Clotting and bleeding time assay, blood collection and platelet measurement
Tail bleeding was measured as described previously [13]. Briefly, anesthetized mice's tail amputated with a sharp scalpel, and bleeding time was then determined by monitoring the duration of animal tail bleeding until it ceased and was kept in a prone position and immersed in PBS. Clotting time measured by the capillary tube method. The mice's tail was cleaned with 70% alcohol and punctured with a 1ml syringe needle. Filled two capillary tubes with free-flowing blood from the puncture site after wiping the first drop of blood. Stop clock started and capillary tubes were broken to see whether a thin fibrin stand formed between two broken ends. After fibrin stand is observed, clotting time measured from the average of two capillary tubes. For platelet measurement, blood obtained by cardiac puncture and collected in EDTA coated MiniCollect tubes (Greiner Bio-One Gmbh, kremsmünster, Austria) as described [13]. The whole blood was used to measure total and of active Platelet count and was carried out through flow cytometry using FACSCalibur (BD Biosciences, USA). Briefly, diluted whole blood (1:4) in PBS was incubated with APC conjugated anti-CD62P (eBioscience Inc, San Diego, CA, USA) and FITC conjugated anti-CD41 (eBioscience Inc, San Diego, CA, USA) for 15 min. Matched fluorescein-conjugated isotype control antibodies were used simultaneously for staining for comparison. The activity was compared using CellQuest Pro software (BD Biosciences, USA).
Bronchoalveolar lavage fluid collection and histopathology
Bronchoalveolar lavage fluid (BAL) was collected by instilling 1 ml PBS into the tracheotomised airway and recovered BAL fluids were processed to get total leukocyte count, as described previously (). For lung histology, the lungs were excised and fixed in 10% buffered formalin. The fixed, paraffin-embedded tissues cut into 5um sections and either stained with hematoxylin and eosin (H&E) to asses inflammation or Masson’s trichome (MT) staining to assess collagen content.
Cecum ligation puncture (CLP) procedure
Mice were anesthetised by injecting intraperitoneally a solution of 1:1 ketamine (75mg/kg) and xylazine (15mg/kg). The abdomen was shaved, and the peritoneum area was disinfected betadine solution followed by wiping with a 70% alcohol. Under aseptic conditions, a 1 cm midline incision was made, and the cecum carefully exposed with the adjoining intestine. The cecum was then tightly ligated with a 3.0 Mersilk (PROLENE, 8680G; Ethicon) sutures at the base and punctured once with a 19-gauge needle on the same side of the cecum. A small amount of stool extruded to ensure patency of the puncture sites. The cecum then returned to the peritoneal cavity, and the wound was closed with 3.0 Mersilk sutures. Control mice (i.e. Sham), the cecum was exposed out and then returned to the peritoneum without ligation or puncture. Mice were resuscitated by injecting subcutaneously 1 ml of pre-warmed 0.9% saline solution using a 25G needle. After surgery, animals placed immediately to a cage with exposure to a heating lamp of 150W until they recovered from the anaesthesia. The recovery time is from 30 min to 1 hour. Mice were monitored every 12 hours for survival or euthanised after 20 hours (n=3) for measurement of cytokines while they were fed with their regular diet and water. Two independent experiments recorded the mortality of mice after CLP surgery.
TGF-b, IL-6,IFN-g, HIF-1a and vWF measurement
The levels of TGF-b, IL-6, IFN-g (BD, USA), HIF-1a (R&D, USA) and vWF (USCN, China) were measured in lung tissue homogenate or in plasma of the mice by sandwich ELISA, as per manufacturer’s protocol.
RNA isolation and whole transcriptome analysis:
Total RNA was isolated from mouse lung tissue treated with AV (AVD2 and AV-D4) or distilled water (vehicle) using the RNeasy Plus Mini Kit (Qiagen, CA, USA) following the manufacturer's protocol. For genome-wide expression analysis, the Affymetrix GeneChip MTA 1.0 array was used according to the manufacturer's instruction. For each sample, 250 ng of RNA was quantified and hybridized to microarray chips following a series of consecutive steps described in the protocol. After hybridization, microarray chips are then scanned using an Affymetrix GCS 3,000 scanner (Affymetrix, CA, USA) and the signal values are further evaluated using the Affymetrix® GeneChip™ Command Console software. Raw data automatically extracted using the Affymetrix data extraction protocol in the Affymetrix GeneChip® Command Console® Software (AGCC). CEL file import, mRNA level, all analysis, and export of the results were all performed using Affymetrix® Expression Console™ software. A comparative study between the vehicle and the AV treated samples done by using fold-change and p-value, genes considered to the differentially expressed by applying the criteria of significance p-value less than or equal to 0.05.
Functional enrichment and Connectivity map analysis
For functional analysis, we used Enrichr (amp.pharm.mssm.edu) tool. For pathway and gene ontology analysis, we examined gene enrichment in Cellular Compartment, Biological Processes, BioPlanet, Wiki, KEGG human pathway and gene set enrichment was considered if P-value less than 0.05 in Enrich r tool. For connectivity map (CMap) analysis, differentially expressed genes ranked according to fold change and list of top 150 up and down-regulated genes compatible with the CMAP data signatures was used to query the connectivity using clue.io touchstone database.
SARS-CoV-2 transcriptome meta-analysis
We obtained the raw RNA sequencing data from SARS-CoV-2 patients Broncho-alveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) from the authors [18] and analysed them inhouse. Functional enrichment and gene set enrichment was done as described above using the differentially expressed genes. The results were then compared with AV transcriptome data to find intersections between gene ontologies, enriched gene sets, and connectivity map perturbations between upregulated genes of BALF, PBMC and downregulated genes of AV and between downregulated genes of BALF, PBMC and upregulated genes of AV.
Molecular docking
The complete genome sequence of the novel SARS-CoV-2 virus was obtained from the National Centre for Biotechnology Information (NCBI) nucleotide database (NC_045512.2). The available 3D crystal structures of all the target proteins such as 3CLpro, PLpro, RdRp, S-protein, ACE2 and JAK2 were taken from protein data bank [19]. Others structures (NSP4, NSP7, NSP8, NSP9, NSP13, NSP14, NSP15 and NSP16, and TMPRSS2) were built using homology modeling with suitable templates using Swiss model [20] and I-TASEER web-servers [21]. The active regions of the proteins were identified by COACH meta-server and the results were compared with results from CASTp web server [22]. The anti-COVID-19 activity of the compounds extracted from the Adhatoda vasica were investigated using Molecular Docking studies using Schrodinger suite (Maestro) [23] and AutoDock vina packages [24]. In Schrodinger suite, all the target proteins were prepared using protein preparation wizard that included optimization followed by minimization of heavy atoms of proteins. The energy minimized 3D structures of all the ligands were prepared using LigPrep. The best pose of ligands that fit well in the protein cavity was carried out using OPLS3 force field with Glide package in Extra Precision mode (XP) mode. According to the size of binding cavity of the proteins, the coordinates x, y and z of the grid box were chosen with the grid resolution of 1 Å for calculations using AutoDock vina package.
Statistical analysis
Statistical significance determined by one-way analysis of variance and analysis was done using GraphPad Prism software. In the case of mice experiment, all data represent mean ± SEM; n= 3-10 in each group and significance denoted by *p <0.05, **p <0.01, ***p <0.001. p-value > 0.05 is considered non-significant (NS). Significance of the survival study determined by Log-rank (Mantel-Cox) test using GraphPad Prism software.