A novel anti-inflammatory treatment for bradykinin-induced sore throat or pharyngitis

Abstract

Background: Often thought of as a minor health concern, sore throat or pharyngitis is an important public health issue. It is one of the most common symptoms of upper respiratory diseases including COVID-19 and is a leading cause of physician visits and antibiotic prescriptions. However, few over-the-counter medications are proven to heal sore throat inflammation.

Methods: Adenocarcinomic human alveolar basal epithelial cells (A549 cells) and three dimensional organotypic human respiratory tissues were used to study inflammation and various treatment effects on respiratory epithelia. The cells and tissues were studied both in the presence and absence of bradykinin, one of the first inflammatory mediators of pharyngitis. Inflammation was measured by analyzing the levels of prostaglandin E2 (PGE2), interleukin 8 (IL-8), and leukotriene B4 (LTB4), transepithelial electrical resistance (TEER), and lactate dehydrogenase (LDH) release. Tissue morphology was analyzed by immunohistochemistry.

Results: In studying pharyngitis using organotypic human respiratory tissue stimulated with bradykinin, we saw an increase in PGE2 and interleukin-8 (IL-8) in response to bradykinin. Acetyl salicylic acid (ASA), a nonspecific COX inhibitor, was able to mitigate a bradykinin-induced increase in PGE2 in our studies. However, ASA was inflammatory above its therapeutic window, increasing the levels of PGE2 and IL-8 above those seen with bradykinin stimulation alone. We describe a novel, scientifically validated treatment for sore throat, that contains a low dose of ASA and other anti-inflammatory ingredients.

Conclusion: This study elucidates the complex mechanisms involved in healing pharyngitis, an inflammatory condition of the upper respiratory epithelia. An ASA-based formula (Biovanta) mitigated bradykinin-induced inflammation more strongly than ASA alone in organotypic human respiratory tissues. Surprisingly, we found that many of the most common over the counter sore throat therapies exacerbate inflammation and IL-8 in organotypic human respiratory tissues, suggesting these common treatments may increase the likelihood of further respiratory complications.

Keywords: Bradykinin; Epiairway; MucilAir; acetyl salicylic acid; pharyngitis; prostaglandin.

© 2021 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.

Figures

Figure 1

(A−C) A549 cells were treated with culture media (DMEM), or the indicated concentrations of bradykinin (BK) in 96‐well microtiter plates. PGE2 levels in media were measured at 4 h postinoculation. IL‐8 was measured 24 h post inoculation (B). LDH levels were measured 24 h postinoculation (C). (D–I) A549 cells were treated with cell culture medium (black bars) or 10 µM of BK (gray bars) 30 min after treatment with various concentrations of ASA or 100 µM NS‐398. PGE2 was measured at 4 h postinoculation (D). IL‐8 was measured 24 h (E) or 48h  (F) postinoculation. LTB4 was measured 4 h (G) and 24 h (H) postinoculation. LDH was measured 48 h postinoculation (I). Mann−Whitney statistical tests were performed using GraphPad Prism software. *p < .05, **p < .01, ***p < .001, and ****p < .0001. ASA, acetyl salicylic acid; DMEM, delbecco's modified eagle medium; IL‐8, interleukin‐8; LDH, lactate dehydrogenase; PGE2, prostaglandin E2

Figure 2

MucilairTM respiratory tissues suspended in transwell inserts were treated with either saliva buffer (vehicle, black bars), or 47 mM bradykinin (gray bars) following treatment with various concentrations of ASA, 100 µM NS‐398, or an ASA‐based formula containing 3 mM ASA (Biovanta liquid). Levels of PGE2 in basal media were measured at 4 h postinoculation (A). IL‐8 levels in basal media were measured at 24 h (B) and 48 h (C) postinoculation. Transepithelial membrane resistance was measured at 24 h (D) and 48 h (E) postinoculation (C). Statistics shown are results of unpaired t test using GraphPad Prism software. Statistics were performed on raw data without omissions and not on fold‐change values. The figures in D are reprinted with permission from Epithelix Sarl. *p < .05, **p < .01, and ***p < .001. ASA, acetyl salicylic acid; PGE2, prostaglandin E2

Figure 3

MucilairTM respiratory tissues suspended in transwell inserts in 24 well plates were treated with saliva buffer (vehicle), bradykinin (BK) (47 mM) or BK following treatments with 100 µM NS‐398 or various over the counter sore throat products. The products are indicated on the graph by their first active ingredient. The key lists all the active ingredients and the trade name of the product. Levels of PGE2 in basal media were measured at 4 h postinoculation (A). IL‐8 levels and LDH in basal media were measured at 24 h (B and D) and 48 h (C and E) postinoculation, and transepithelial electrical resistance was measured at 24 h (F) and 48 h (G) post inoculation. Statistics shown are results of ANOVA and unpaired t tests using GraphPad Prism software. Statistical analysis was performed on raw data without omissions and not on fold‐change values. *p < .05, **p < .01, ***p < .001, and ****p < .0001. ANOVA, analysis of variance; IL‐8, interleukin‐8; LDH, lactate dehydrogenase; PGE2, prostaglandin E2

Figure 4

EpiAirwayTM (AIR‐200‐PE6.5) respiratory tissues suspended in transwell inserts were treated with saliva buffer (vehicle) and various sore throat treatments as indicated. The products are indicated on the graph by their first active ingredient. The key lists all the active ingredients and the trade name of the product. Measurements of PGE2 at 4 h (A), IL‐8 at 24 h (B), LDH at 24 h, (C) and TEER at 24 h (D) are shown. Statistics shown are results of Kruskal–Wallis tests performed using GraphPad Prism software. Statistics were performed on raw data without omissions and not on fold‐change values. **p < .01. IL‐8, interleukin‐8; LDH, lactate dehydrogenase; PGE2, prostaglandin E2; TEER, transepithelial electrical resistance

Figure 5

This study was performed in a third party blind format. EpiAirwayTM (AIR‐200‐PE6.5) respiratory tissues suspended in transwell inserts were either left untreated, treated with saliva buffer (vehicle), triton‐X (positive control), or various sore throat products as indicated. The various products are indicated on the graph by their first active ingredient. The key lists all the active ingredients and the trade name of the product. Following their respective treatments, all tissue inserts (except the untreated) were treated with 15 mM bradykinin. TEER was measured 48 h after treatment. Statistics shown are results of a Kruskal–Wallis test using GraphPad Prism software, ****p < .0001. Statistics were performed on raw data without omissions and not on fold‐change values. TEER, transepithelial electrical resistance

Figure 6

Histological analysis of the tissues analyzed in the third‐party blind study, a representative sample of the tissues used in Figure 5 are shown. In all of the tissues, except those treated with saliva buffer (vehicle), bradykinin, or the ASA lozenge (Biovanta lozenge), marked histological damage was seen in response to treatment. After fixation and embedding in parrafin, sections were cut into 5 um thick section and stained with hemotoxylin and eosin. ASA, acetyl salicylic acid