Application of Humanized Zebrafish Model in the Suppression of SARS-CoV-2 Spike Protein Induced Pathology by Tri-Herbal Medicine Coronil via Cytokine Modulation

Abstract

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV-V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1β induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.

Keywords: Ayurveda; Coronil; SARS-CoV-2 infection; anti-inflammatory; behavioral fever; herbal medicine; humanized zebrafish; swim bladder.

Figure 1

UHPLC analysis identified the presence of bio-active ingredients in Coronil. The Coronil sample (purple lines) was crushed into a fine powder and analyzed by UHPLC using reference standards mix (blue lines). The chromatographs were recorded at 210 nm (for betulinic acid and ursolic acid), 227 nm (for withanone, withaferine A, withanoside IV, withanoside V, cordifolioside A and magnoflorine), and 325 nm (for rosmarinic acid and palmatine) wavelengths. The chemical structures of these identified compounds, retention time (in minutes), concentration (µg/mg) and the plant species it was derived from are given in Table 1.

Figure 2

Study design of SARS-CoV-2 spike protein induced pathology using humanized xeno-transplanted zebrafish. (a) Representative images of swim bladders from (i) NCTL and (ii) HZFCTL post 7-day transplant showing normal morphological traits. Cyto-smears of (iii) NCTL and (iv) HZFCTL showed normal cytological features along with transplanted cells (zoom-in represents xeno-transplanted cells). Black arrows indicate cells of zebrafish origin and black circle represent cells of human origin. AL—anterior lobe, PL—posterior lobe. Scale bars, 1000 μm at 1X magnification. (b) Schematic representation of zebrafish model of SARS-CoV-2 spike protein induced pathology. Humanized zebrafish (n = 24 in each group) were injected with SARS-CoV-2 spike protein (Disease Control, DCTL) or PBS (Normal Control, NCTL and Humanized zebrafish control, HZFCTL) and treated with two different doses of Coronil, 12 μg/kg (CN12) and 58 μg/kg(CN58) and DEX (0.08 μg/kg) for two time points followed by necropsy and endpoint measurement. (c) Kaplan-Meier survival curves following SARS-CoV-2 spike protein challenge and pharmacological treatment with DEX and Coronil till 10th day. n = 24. Log rank test ** p < 0.001.

Figure 3

Coronil reduces SARS-CoV-2 spike protein induced behavioral fever. Heat map of time spent (in seconds) at the set temperatures (low temperature 23 °C, normal temperature 29 °C, and behavioral fever 37 °C) by the zebrafish from various groups on the fourth and seventh day time points. Color intensity indicates the time spent at the particular temperature chamber. SARS-CoV-2 spike protein induced behavioral fever was decreased in a dose dependent manner upon CN treatment at the seventh day time point.

Figure 4

Coronil reduces SARS-CoV-2 induced swim bladder structural abnormality and edema. (Normal control at 4th day (a), HZFCTL at 4th day (b) and NCTL at 7th day (g), HZFCTL at 7th day (h)) NCTL and HZFCTL at the 4th and 7th day time points, respectively, showing normal swim bladder. (c,i) SARS-CoV-2 spike protein induced structural anomaly and edema. (d) DEX treatment at the 4th day time point showed mildly inflated swim bladder. (j) DEX treatment at the 7th day time point showed narrowing of the posterior lobe due to edema. (e,f) At the 4th day time point, the CN12 and CN58 groups were comparable to the NCTL and HZFCTL groups. (k,l) At the 7th day time point, the CN12 and CN58 groups were comparable to NCTL. n = 24 for each test group, AL—anterior lobe, PL—posterior lobe. Circle inside the swim bladder indicates the watermark at the contact between the glass slide and swim bladder during the image capturing process. Scale bar, 1000 μm at 1X magnification.

Figure 5

Coronil rescues from SARS-CoV-2 spike protein induced inflammation in swim bladder cytosmears. Cytosmears of the swim bladder were analyzed for inflammatory cell accumulation based on cell and nuclear morphology. Representative images of Hematoxylin and Eosin (H&E) stained swim bladder cytosmears of (a) NCTL at 4th day and (g) NCTL at 7th day and HZFCTL at 4th day (b) and at 7th day (h) showed cells with normal morphological characteristics. (c,i) DCTL showed infiltration of granulocytes and macrophages and cellular materials and nuclear debris and edema. (d,j) DEX treatment rescued from inflammation along with the presence of cellular debris. (e) Coronil (12 µg/kg) at 4th day, (f) Coronil (58 µg/kg) at 4th day; (k) Coronil (12 µg/kg at 7th day, and (l) Coronil (58 µg/kg at 7th day. Dose dependent rescue of cytological disease markers on both the 4th day (e—CN12, f—CN58) and 7th day (k—CN12, l—CN58) time points upon Coronil treatment as indicated by the presence of epithelia and myocytes along with lymphocyte and macrophage aggregates and reduced inflammatory cellular debris. (n) Quantitative enumeration of granulocytes using swim bladder cyto-smear. (o) SARS-CoV-2 induced macrophage infiltration was significantly decreased upon CN treatment at the 4th day time point. (p) Lymphocyte infiltration was significantly increased upon DEX or CN treatment at both time points. Scale bars, 10 μm at 200X magnification. n = 24 for each test group. ## represents statistical significance compared to HZFCTL and ** represents significant compared to DCTL (p < 0.001). E—epithelial nuclei; S—smooth muscle cell nuclei; M—melano-macrophage. Black arrow—normal cell, yellow arrow—lymphocyte, brown arrow—macrophage and green arrow—granulocyte.

Figure 6

Gross anatomy of kidney to test the effect of SARS-CoV-2 spike protein and Coronil on renal architecture. (a) NCTL at 4th day, (b) HZFCTL at 4th day, (g) NCTL at 7th day, (h) HZFCTL at 7th day. (c,i) DCTL with lost tubular segmentation and vascular degeneration. (d,j) DEX. (e,f) CN12 and CN58 of 4th day group exhibiting normal renal morphology. (k) CN12 showed normal renal architecture at the 7th day time point. (l) CN58 exhibiting mildly degenerative renal pathology. H—Head; S—Saddle; T—Tail of kidney. n = 24 for each test group, scale bars, 1000 μm at 1X magnification.

Figure 7

Coronil rescues from SARS-CoV-2 spike protein induced renal cell damage. Cytosmears of whole kidney necropsies were made and stained with H&E. The average degenerative and necrotic nuclei per 3 field were quantified to study renal pathological features at both the 4th and 7th day time points. (a) NCTL at 4th day, (b) HZFCTL at 4th day, (g) NCTL at 7th day, (h) HZFCTL at 7th day. NCTL and HZFCTL with apparent cytological features without any necrotic and degenerating cells. (c,i) DCTL with necrotic and degenerating renal cells. (d,j) DEX group. (e) Coronil (12 µg/kg) at 4th day, (f) Coronil (58 µg/kg) at 4th day; (k) Coronil (12 µg/kg at 7th day, and (l) Coronil (58 µg/kg at 7th day. CN12, CN58 at 4th day and 7th day time points. (m) Renal cell degeneration was significantly decreased in a dose dependent manner at the 7th day time point. (n) Spike protein induced renal cell necrosis % was significantly decreased at the 4th day time point. Black arrow—normal cell, Red arrow—necrotic cell and Yellow arrow—degenerating cell. Scale bars, 10 μm at 200X magnification. n = 24 for each test group. ## represents statistical significance compared to HZFCTL and ** represents significance compared to DCTL (p < 0.001).

Figure 8

Pharmacological treatment with Coronil inhibits SARS-CoV-2 spike protein induced hemorrhage. Zebrafish were euthanized at end of study and whole animal imaging was performed from all groups and analyzed for spike protein induced skin hemorrhage on the 4th day and 7th day time points. (a) NCTL at 4th day, (b) HZFCTL at 4th day, (g) NCTL at 7th day, (h) HZFCTL at 7th day. (c,i) DCTL showing skin hemorrhage circled in yellow. (d,j) DEX group. (e) Coronil (12 µg/kg) at 4th day, (f) Coronil (58 µg/kg) at 4th day; (k) Coronil (12 µg/kg at 7th day, and (l) Coronil (58 µg/kg at 7th day. (m) Qualitative analysis of the number of fish with hemorrhage across each treatment group and respective time points. (n) Quantitative analysis of area of hemorrhage. n = 24 for each group, ## represents statistical significance compared to HZFCTL and ** represents significance compared to DCTL (p < 0.001).

Figure 9

Coronil decreases the pro-inflammatory responses in vitro. A549 cells were treated with various concentrations of CN for 24 h and measured for toxicological profile. (a) Cytotoxicity of CN on A549 cells expressed as % cell viability. Cells were pre-treated with CN followed by co-treatment with CN + IL-1β for 24 h and assayed for the secretion of cytokines using Enzyme Linked Immunosorbent Assay (ELISA). Secretion of (b) IL-6 and (c) TNF-α. (d) HEK Blue TNF-α reporter Secreted Alkaline phosphatase (SEAP) assay for NF-κB/AP-1 transcriptional activity. HEK blue cells stably expressing the NF-κB/AP-1-SEAP reporter gene were pre-treated with various doses of CN followed by treatment with TNF-α and measuring SEAP activity after 24 h. Data are presented as the means ± SEM (n = 2 in triplicates). **, ^##, p < 0.001 by one-way ANOVA; ^## represents significance compared to NC and ** represents significance compared to IL-1β.

Figure 10

Schematic representation of the experimental tank setup with a variation of three temperature (23, 29 and 37 °C) gradient, interconnected chambers for studying behavioral fever in a study of zebrafish.