Inhibition of SARS-CoV-2-Induced NLRP3 Inflammasome-Mediated Lung Cell Inflammation by Triphala-Loaded Nanoparticle Targeting Spike Glycoprotein S1

Abstract

The COVID-19 pandemic, caused by SARS-CoV-2, poses a significant global health threat. The spike glycoprotein S1 of the SARS-CoV-2 virus is known to induce the production of pro-inflammatory mediators, contributing to hyperinflammation in COVID-19 patients. Triphala, an ancient Ayurvedic remedy composed of dried fruits from three plant species-Emblica officinalis (Family Euphorbiaceae), Terminalia bellerica (Family Combretaceae), and Terminalia chebula (Family Combretaceae)-shows promise in addressing inflammation. However, the limited water solubility of its ethanolic extract impedes its bioavailability. In this study, we aimed to develop nanoparticles loaded with Triphala extract, termed "nanotriphala", as a drug delivery system. Additionally, we investigated the in vitro anti-inflammatory properties of nanotriphala and its major compounds, namely gallic acid, chebulagic acid, and chebulinic acid, in lung epithelial cells (A549) induced by CoV2-SP. The nanotriphala formulation was prepared using the solvent displacement method. The encapsulation efficiency of Triphala in nanotriphala was determined to be 87.96 ± 2.60% based on total phenolic content. In terms of in vitro release, nanotriphala exhibited a biphasic release profile with zero-order kinetics over 0-8 h. A549 cells were treated with nanotriphala or its active compounds and then induced with 100 ng/mL of spike S1 subunit (CoV2-SP). The results demonstrate that chebulagic acid and chebulinic acid are the active compounds in nanotriphala, which significantly reduced cytokine release (IL-6, IL-1β, and IL-18) and suppressed the expression of inflammatory genes (IL-6, IL-1β, IL-18, and NLRP3) (p < 0.05). Mechanistically, nanotriphala and its active compounds notably attenuated the expression of inflammasome machinery proteins (NLRP3, ASC, and Caspase-1) (p < 0.05). In conclusion, the nanoparticle formulation of Triphala enhances its stability and exhibits anti-inflammatory properties against CoV2-SP-induction. This was achieved by suppressing inflammatory mediators and the NLRP3 inflammasome machinery. Thus, nanotriphala holds promise as a supportive preventive anti-inflammatory therapy for COVID-19-related chronic inflammation.

Keywords: NLRP3 inflammasome pathway; SARS-CoV-2; anti-inflammation; nanoparticles; nanotriphala.

Figure 1

Physical characteristic of nanotriphala, (A) particle size, (B) polydispersity index, and (C) zeta potential of nanotriphala at various storage times (0.25, 1, 2, and 3 months) and temperatures (4 °C, 30 °C, and 45 °C). Particle size and polydispersity index were determined using dynamic light scattering, while zeta potential values were analyzed by measuring the electrophoretic mobility of nanotriphala. Data are presented as mean ± S.D. from three independent experiments. Statistical significance compared to freshly prepared nanotriphala is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 2

Folin–Ciocalteu assay standard curve. A standard curve depicting the relationship between concentration and absorbance in the Folin–Ciocalteu assay. The concentration of gallic acid equivalent was determined using the equation y = 0.0136x + 0.0727, with an r² of 0.9998, where y represents absorbance and x represents the concentration of gallic acid. In the standard reaction, 50 µL of gallic acid concentration was mixed with 100 µL of Folin–Ciaocalteu phenol reagent and left to incubate at room temperature for 2 h. Then absorbance was subsequently measured at 765 nm. The resulting calibration curve was used to calculate the total phenolic content in nanotriphala.

Figure 3

In vitro release profile of phenolic contents from nanotriphala. The graph depicts the in vitro release profile of phenolic contents from nanotriphala in phosphate buffered solution at pH 6.5 and 37 °C. Samples of nanotriphala were collected at various time intervals: 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h. The total phenolic content was analyzed using the Folin–Ciocalteu method.

Figure 4

Long-term chemical stability prediction of nanotriphala. The figure illustrates the long-term chemical stability prediction of nanotriphala, extrapolated from accelerated stability data over a 3-month period of storage at temperatures of 4, 30, and 45 °C. The total phenolic content was quantified using the Folin–Ciocalteu method. Analysis of the determination coefficient (r²) indicated that the chemical degradation of nanotriphala followed zero-order kinetics.

Figure 5

Cell viability of nanotriphala and blank control (A), Triphala extract (B), gallic acid (C), chebulagic acid (D), and chebulinic acid (E) on A549 cells. Cell viability of nanotriphala and blank control (F), Triphala extract (G), and gallic acid (H) on primary human dermal fibroblasts. Cells were treated with blank control, nanotriphala (0–300 μg/mL), and active compounds (gallic acid, chebulagic acid, and chebulinic acid) (0–50 μg/mL) for 24 and 48 h. Cell survival was determined using an MTT assay. Data are presented as mean ± S.D. values of three independent experiments, *** p < 0.001 compared with the control group.

Figure 6

Inhibitory effects of nanotriphala and blank control on the pro-inflammatory cytokine secretion in CoV2-SP-induced A549 cells. A549 cells were pre-treated with nanotriphala and blank control (0–300 µg/mL) for 24 h. Then, the cells were exposed to CoV2-SP (100 ng/mL) for 3 h. The IL-6 (A), IL-1β (B), and IL-18 secretions (C) in the culture supernatant were examined by ELISA. The CoV2-SP-induced A549 cells are presented as 100%. Data are presented as mean ± S.D. values of three independent experiments, *** p < 0.001 compared with the CoV2-SP-induced control group. ^a * p < 0.05, ^a ** p < 0.01, ^a *** p < 0.001 compared with blank control at the same concentration.

Figure 7

Inhibitory effects of active compounds (gallic acid, chebulagic acid, and chebulinic acid) on the pro-inflammatory cytokine secretion in CoV2-SP-induced A549 cells. A549 cells were pre-treated with active compounds (gallic acid, chebulagic acid, and chebulinic acid) (0–20 µg/mL) for 24 h. Then, the cells were exposed to CoV2-SP (100 ng/mL) for 3 h. The IL-6 (A), IL-1β (B), and IL-18 secretions (C) in the culture supernatant were examined by ELISA. The CoV2-SP-induced A549 cells are presented as 100%. Data are presented as mean ± S.D. values of three independent experiments, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the CoV2-SP-induced control group. ^a p < 0.001 compared with gallic acid at the same concentration.

Figure 8

Inhibitory effects of nanotriphala and blank control on the IL-6 (A), IL-1β (B), IL-18 (C), and NLRP3 gene expressions (D) in CoV2-SP-induced A549 cells. A549 cells were pre-treated with nanotriphala and blank control (0–300 µg/mL) for 24 h. Then, the cells were exposed to CoV2-SP (100 ng/mL) for 3 h. The mRNA expressions were determined using RT-qPCR. Data are presented as mean ± S.D. values of three independent experiments, ** p < 0.01 and *** p < 0.001 vs. the CoV2-SP-induced control group. ^a * p < 0.001 < 0.05, ^a ** p < 0.01, ^a *** p < 0.001 compared with blank control at the same concentration.

Figure 9

Inhibitory effects of active compounds (gallic acid, chebulagic acid and chebulinic acid) on the IL-6 (A), IL-1β (B), IL-18 (C), and NLRP3 gene expressions (D) in CoV2-SP-induced A549 cells. A549 cells were pre-treated with active compounds (gallic acid, chebulagic acid and chebulinic acid) (0–20 µg/mL) for 24 h. Then, the cells were exposed to CoV2-SP (100 ng/mL) for 3 h. The mRNA expressions were determined using RT-qPCR. Data are presented as mean ± S.D. values of three independent experiments, * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the CoV2-SP-induced control group. ^a p < 0.001 compared with gallic acid at the same concentration.

Figure 10

The effects of nanotriphala inhibited the NLRP3 inflammasome pathway in CoV2-SP-induced A549 cells. A549 cells were pre-treated with nanotriphala (0–300 µg/mL) for 24 h. Then, the cells were exposed to CoV2-SP (100 ng/mL) for 3 h. The inhibitory effects of nanotriphala on the expression of NLRP3, ASC, and pro-caspase-1 (p50) and cleaved-caspase-1 (p20) proteins in A549 cells are displayed in western blot (A) and band density measurements (B). The CoV2-SP-induced A549 is presented as 100% of control. Data are presented as mean ± S.D. values of three independent experiments, ** p < 0.01, and *** p < 0.001 compared with the CoV2-SP-induced A549 cells.

Figure 11

The effects of chebulagic acid and chebulinic acid inhibited the NLRP3 inflammasome pathway in CoV2-SP-induced A549 cells. A549 cells were pre-treated with chebulagic acid and chebulinic acid (0–20 µg/mL) for 24 h. Then, the cells were exposed to CoV2-SP (100 ng/mL) for 3 h. The inhibitory effects of chebulagic acid and chebulinic acid on the expression of NLRP3, ASC, and pro-caspase-1 (p50) and cleaved-caspase-1 (p20) proteins in A549 cells are displayed in western blot (A,B) and band density measurements (C,D). The CoV2-SP-induced A549 is presented as 100% of the control. Data are presented as mean ± S.D. values of three independent experiments, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the CoV2-SP-induced A549 cells.