INTRODUCTION:

TLRs, a subclass of host defense receptors, are thought to play a crucial role in the early stages of the inflammatory responses brought on by several antigenic pathogens¹. TLR-3 is a motif recognition receptor found in mammals that identify dsRNA in addition also synthesized dsRNA analog polyriboinosinic ribocytidylic acid². In-vivo long-term TLR3 expression is thereby considered to be an inflammation brought caused by necrosis or viral agents³. Additionally, our results demonstrate that TLR3 ligation activates transcriptional processes and phosphorylation, which produces a range of inflammatory proteins hypothesized may support the innate immune system⁴. Overall, our findings show that prolonged TLR3 activation can be an essential factor in the regulation of inflammatory disorders.Asthma exacerbations are frequently caused by viral infections^5,6.

Bacterial infections can potentially worsen these illnesses. TLRs are at the frontline in microbial resistance, with TLR3 reacting to viral activation. The existing work intended the efficacy of a new TLR3 /dsRNA fusion inhibitor (R)-2-(3-Chloro-6-fluorobenzo[b] thiophene-2-carboxamido)-3-propanoic acid inside a mice model for allergic asthma⁷. Exacerbations in lung diseases, like obstructive lung illness (COPD), are indicated by greater symptoms and even a loss of bronchial function⁸. The production of the lungs' pro-inflammatory chemokines upon viral exposure is a vital turn in establishing the signal of inflammatory activity in some lung illnesses⁹. Viruses such as paramyxovirus and human rhinovirus are suspected of causing viral asthma exacerbations. In asthma patients, bacterial and viral infiltrations coexist. In allergic mice, respiratory virus infections worsen Th2 diseases. Infection with the influenza virus promotes the spread the lung diseases, resulting in higher Th2 activity¹⁰. This investigation utilized an OVA-induced asthma mice model of viral bronchial inflammation¹¹. The effects of TLR3 activation-induced indigenous dsRNA retroviruses on lung inflammation are investigated in mice by sensitization and challenge via OVA and Poly (I.C). We looked at changes in oxidative stress markers, AHR and animals’ body mass. Inhibiting the TLR-3/dsRNA complex restricts dsRNA from binding to TLR-3, which is then linked to innate immune abnormalities in bronchial viral inflammation.

MATERIALS AND METHODS:

Animals and Reagents:

Swiss albino mice were acquired from the AIIMS Central Animal House in New Delhi, India, and kept in environments free of pathogens at 20–25°C and 50–70 % humidity. During the week preceding an experiment, ad libitum access to tap water and an OVA-free diet was offered. All other analytical-grade substances arose from Sigma Chemicals acquisitions, St. Louis, Missouri, USA. Ovalbumin (CAS No. 138831-86-4 and Cat no. S7951), TLR3 agonist, Poly (I: C) (CAS no. 31852-29-6 and Cat no. S7951), TLR3/dsRNA complex inhibitor Calbiochem (Cat no. - 614310), Dexamethasone (CAS No. 50-02-2 and Cat no. D1756) purchased from Thermo-Fisher Scientific, USA.

Study Protocol:

The mice were placed into seven groups of six individuals each, and they received therapy for 22 days. 24hrs after the last medication, the animals were weighed, killed, and the lung lobes were evacuated and cleansed using the usual saline solution. The doses of Ova (OC) (10mg/kg i.p./50mg, i.n.) in 1mg Al(OH)3 (Sigma-Aldrich), Poly (I.C) (40mg/kg i.p./20mg i.n.), TLR-3/dsRNA complex antagonist (1mg/kg/, 3mg/kg and 5mg/kg, i.p.) and dexamethasone (1mg/kg i.p.) dissolved in PBS which is used as a vehicle^7,12–14

Measurement of Antioxidant markers:

A homogenate that had been homogenized with 1.15% KCl at a ratio of 1:10 was the subject of an MDA study on 50% of the homogenate. The supernatants from a different section that was centrifuged for more than an hour at 5000g (at +4°C) were also collected, and assays for GPx, CAT, and GSH were run¹⁵. This method led to the discovery of the decreased GSH concentration. Nanomoles per milliliter (nmol/ml) is the unit of measurement for MDA and GSH concentrations¹⁶. The Aebi (1984) method has been used to test the activity of the CAT enzyme, while the Beutler (1975) method has been used to access the reactivity of the GPx enzyme (Weissman)¹⁷. The protein screening of the centrifuged and supernatant was done by Lowry et al. technique (1951)¹⁸

Effect of treatments on Body weight:

Body weight measurements: Before administering OVA, Poly (I.C), and TLR3/dsRNA inhibitor, the animal's body mass was measured on days 0, 7, 14, 21, 22, and 23. The study's principal investigator kept close track of basic asthmatic symptoms including reduced activity, general unease, breathing issues, and wheezing throughout to ensure integrity. Although most of the mice had these symptoms right away after using the nebulization approach, they did not significantly hurt the animals and eventually went away. Until the final group of animals got slaughtered on day 23, the weighing continued. The mean weight in (gms) for each group on each dosage day was computed, and the corresponding standard deviation data were reported as mean SEM¹⁹

Statistical Evaluation:

The results are given as mean±S.E.M. The one-way ANOVA and post hoc Tukey's differentiations test were applied for the statistical analyses; data were deemed statistically significant at P<0.05. Statistical calculations were done through Graph pad prism software (San Diego, CA, USA).

RESULTS:

Inhibition of TLR3/dsRNA complex formation lowers Poly (I.C)-induced oxidative stress markers:

As part of an ongoing study to determine how TLR3/dsRNA complex inhibitor affected antioxidant indicators in an asthma rodent model, GSH and MDA levels in lung cells, along with CAT and GPx enzymatic activities, were assessed. Data showed that OVA and Poly (I.C) groups had considerably higher MDA levels than the other groups. On the other hand, it was demonstrated that TLR3/dsRNA inhibitor medication reduced MDA levels to levels equivalent to control group. The reduced MDA rates in the dexamethasone standard group while compared with the control group. When MDA levels were compared across groups, the OVA + TLR3 / dsRNA inhibitor (5mg/kg) and control groups showed non-significant results, but OVA and Poly (I.C) groups showed considerably higher MDA levels. When GSH levels were analyzed within the group, both OVA and Poly (I.C) groups exhibited a substantial decline compared with the control group. The reduction in GSH level, on the other hand, was greatly prevented within OVA+Poly (I.C) +TLR3/dsRNA C.I (5mg/kg and 3mg/kg) and DEX groups (p<0.001) as indicated in table-1. The TLR3/dsRNA inhibitor's effect was somewhat greater than the DEX-treated group. When the GPx enzyme activity was examined, it was found that enzyme activity was considerably lower in the possibly harmful OVA and Poly (I.C) treated group in contrast with the control group. Whereas, GPx enzyme activity was considerably increased within OVA+ Poly (I.C) +TLR3/dsRNA C.I(5 mg/kg) and OVA+Poly(I.C)+DEX (1mg/kg) fed group compared to OVA+ Poly (I.C) groups (p<0.001). No statistical difference existed (p<0.05) when CAT enzymatic activity throughout lung tissue was tested in the group (Figure-1).

Table:1 Antioxidant biomarkers concentration in lung tissues

Groups	MDA (nmol/ml)	GSH (nmol/ml)	GPx (U/g protein)	CAT (U/ml)
NC	4.028 ± 0.68	6.121 ± 0.39	181.521 ± 2.78	35.521 ± 2.19
OC	6.972^## ± 0.79	4.121^# ± 0.57	151.421^# ± 3.71	30.421^ns ± 2.81
OVA+Poly (I.C)	10.854^@ ± 0.88	2.832^ns ± 0.73	121.632^@@ ± 3.67	23.632^@ ± 2.95
OVA+Poly (I.C) +TLR3/ dsRNA C.I (1mg/kg)	9.751^** ± 0.93	3.110^* ± 0.35	141.010^** ± 2.60	28.010^* ± 1.81
OVA+Poly (I.C) +TLR3/ dsRNA C.I (3mg/kg)	7.531^* ± 0.78	4.081^ns ± 0.23	147.981^* ± 2.42	33.781^** ± 1.85
OVA+Poly (I.C) +TLR3/ dsRNA C.I (5mg/kg)	5.141^** ± 0.71	4.841^* ± 0.08	168.541^*** ± 2.02	34.941^* ± 1.93
OVA+Poly (I.C) +DEX (1mg/kg)	5.740^* ± 0.75	4.240^ns ± 0.20	165.140^* ± 2.24	33.840^ns ± 1.97

The values are provided as Mean ± SEM (n=6). The data significance was evaluated through one-way ANOVA then Tukey's multiple data comparison tests were applied. ^###p < 0.001 significant, versus NC; ^@p < 0.05 significant versus OC; ^**p<0.01, ^***p<0.001 significant, versus OVA+Poly(I.C) and ns is non-significant versus OVA+Poly(I.C). MDA: Malondialdehyde, GSH: Reduced glutathione, GPx: Glutathione peroxidase, CAT: Catalase

Figure: 1 Illustrate graphical representation of antioxidant marker concentration in lung tissues at different doses of TLR3/dsRNA C.I (1mg/kg, 3mg/kg, 5mg/kg) after sensitization and challenged by OVA and Poly(I.C).All results were presented as Mean± SEM (n=6). As compare sets of data, one-way ANOVA was employed, preceded by Tukey's test. ^###p < 0.001 significant, versus NC; ^@p < 0.05 significant versus OC; ^**p<0.01, ^***p<0.001 significant, versus OVA+ Poly(I.C) and ns is non-significant.

Table 2: TLR-3/dsRNA antagonist effect on body mass variations

Groups	0^thDay	7^th Day	14^th Day	21^st Day	22^nd Day	23^th Day
NC	27.96 ± 0.72	28.71 ± 0.71	29.16 ± 0.68	29.56 ± 0.69	30.58 ± 0.66	30.93 ± 0.61
OC	26.76* ± 0.61	27.41^ns ± 0.67	25.75** ± 0.59	25.10***± 0.51	24.5** ± 0.52	25.20** ± 0.46
OVA+Poly(I.C)	29.66^## ± 0.56	28.65^# ± 0.58	28.00^### ± 0.59	27.15^ns ± 0.60	27.10^# ± 0.59	26.20^ns ± 0.52
OVA+Poly(I.C)+TLR3/dsRNA C.I (1mg/kg)	29.23^@ ± 0.30	28.75^ns ± 0.36	28.15^ns ± 0.37	28.98^@ ± 0.35	28.08^@@± 0.35	29.25^@ ± 0.28
OVA+Poly(I.C)+TLR3/dsRNA C.I (3mg/kg)	25^@@@ ± 0.47	26^@ ± 0.48	27.61^@@ ± 0.46	26.5^ns ± 0.52	27.5^@ ± 0.51	28.08^ns ± 0.48
OVA+Poly(I.C)+TLR3/dsRNA C.I (5mg/kg)	26.5^ns ± 0.70	27.5^@@± 0.69	27.81^ns ± 0.75	28.46 ± 0.70	28.93^ns ±0.73	29.00^@ ± 0.71
OVA+Poly(I.C)+DEX(1mg/kg)	28.26^@ ± 0.43	26.16^@ ± 0.44	28.76^@@ ± 0.36	28.06^ns ± 0.37	29.86^@ ± 0.26	29.03^@@± 0.39

The values are given in mean SEM (n=6). Swiss albino mice (25g-30g) have been administered into animal groups for 0, 7, 14, 21, 22, and 23 days, with substantial weight increases in the NC and TLR3/dsRNA antagonist groups at the 7th, 14th, 21st, and 23rd days, and weight decreases in the lethal groups (OVA and Poly I.C) at the 14th, 21st, and 23rd days. For determining the significance of data, use two-way ANOVA followed by Bonferroni posttests. ***p < 0.001 very significant vs. NC; ^##p<0.01 significant vs. OC, ^@p < 0.05 less significant vs. OVA+Poly I.C , while ns is non-significant.

Figure-2: After injecting OVA/Poly (I.C)/TLR3/dsRNA C.I/DEX to mice, body weight variation was assessed in NC controls, OC, OVA+Poly (I.C) (40g), and TLR3 /dsRNA complex inhibitor drug at various dosages (1mg/kg, 3mg/kg, and 5mg/kg). In every treatment group contrasted with control group, the percentage mean weights were higher throughout the experiment. These results show that long-term treatment with OVA and Poly (I.C) results in considerably decreased body weight but sustained weight gain when coupled with TLR3/dsRNA C.I (5mg/kg, i.p.) and DEX (1mg/kg, i.p.) doses as opposed to TLR3/dsRNA C.I (1mg/kg).

DISCUSSION:

Being among the most prevalent chronic, noncommunicable diseases, asthma has an impact on people's lives and well-being, placing a strain on families, communities, and nations²⁰. Asthma is reportedly difficult to cure due to the intricacy of its pathology, and it has been noted that understanding the underlying processes is crucial for the development of potent medications with minimal side effects²¹. Herbal therapies are also proven their potential²². The airway irritation caused by the OVA model and the signs and symptoms of allergic asthma in people has numerous similarities²³. The study examined the anti-inflammatory properties of TLR3/dsRNA complex inhibitor (Calbiochem) there in an asthma model caused by OVA. To compare the effectiveness of the antagonist, (R)-2-chlorophenyl propanoic acid, a chemical that inhibits the TLR3/dsRNA complex, was administered in mice together with OVA and Dexa in this case. After experimental application, lung tissue underwent biochemical estimations. Previous research has suggested that asthmatic patients' airways are activated in a way that results in heavy oxidative stress and also increased mucus together with the creation of sputum and injured airway cells²⁴.

The oxidative stress indicators (MDA, GPx, CAT, and GSH) were considerably high in OVA-treated mice than in the control group during the research^25,26. According to reports, exposure to OVA disrupts redox equilibrium and weakens the antioxidant defense system, which causes the generation of extremely reactive hydroxyl radicals, an increase in lipid peroxidation, and cellular damage. However, it was shown that antioxidant marker levels in the group treated with TLR3/dsRNA complex inhibitor fell to those of the control group.

The primary substance released in allergic asthma is IgE, which is generated by B cells and binds to certain FcεRI binding sites on mast cells. This process causes the mast cells to degranulate and release allergic broncho-constrictive substances such as leukotrienes, prostaglandins, and histamine²⁷. Several allergens have been implicated in the pathophysiology of asthma, namely in the development of Th1-Th2-associated immune responses²⁸. When all of the study's findings are considered, it is possible to conclude that a TLR3/dsRNA antagonist chemical may have an impact on the levels of Th1/Th2 cytokines generated via OVA thus stopping the progression of asthma in mice²⁹.

The results from the analysis of body weight alterations and inflammatory indicators are supported by the histological data collected as part of the study. OVA exposure has been linked to interstitial pathological changes, emphysema, as well as mucosal destruction in the lungs of mice, which supports the theory that sensitivity is induced³⁰.

In this work, we showed that TLR3 signaling suppression with a novel TLR3/ dsRNA matrix inhibitor significantly reduced lung inflammatory features. It was demonstrated that the TLR3/dsRNA complex inhibitor (R)-2-chlorophenyl propanoic acid had a protective effect by inhibiting TLR3 downstream signaling via TLR3 at the receptor level³¹.

This study shows that Poly (I.C), which causes asthmatic symptoms in mice, and the TLR3/ dsRNA complex inhibitor initiative suppress the level of oxidative stress markers in lung tissues as well as body mass ratio. The development of such an antiviral asthma exacerbation management strategy may be possible with the help of this new chemical.

CONCLUSION:

With the advancement of antagonistic receptor pathways, new therapeutic options for addressing asthma genesis and aggravation are now possible. In conclusion, our data show that animal asthma infections had higher viral loads than human infections. Through a unique TLR-3/dsRNA complex inhibitor, an independent route, Poly (I.C) activation worsens bronchial inflammation in Swiss mice that have been exposed to OVA. In the end, our research revealed that the TLR-3/dsRNA fusion inhibitor and DEX standard treatment groups gradually increased mouse weight as well as improved lung AHR. According to findings, a new TLR3/dsRNA complex inhibitor appears to be a workable drug for development in antiviral asthma exacerbation management and may be a viable target for remedies to respiratory viral infections.

ABBREVIATIONS:

BALF - Bronchoalveolar Lavage Fluid, DEX – Dexamethasone, dsRNA- Double-stranded RNA, OVA – Ovalbumin, Poly (I: C)- Polyinosinic: polycytidylic acid, TLR – Toll-Like Receptor

CONFLICT OF INTEREST:

The authors have no conflict of interest regarding this study.

ACKNOWLEDGMENTS:

The authors are grateful to Amity University, Noida, for assisting with internet access, journal access, and research supervision. We also thank the R.V. Northland Department of Pharmacy for providing the necessary research facilities for the experimental study.

ETHICAL STATEMENT:

To verify the validity of the experimental findings, only 6 mice per group were used per ethical standards, and the tests were conducted by methods approved by R.V. Northland Institute's Animal Institutional Ethical Committee (no. 1149/PO/Re/S/07/CPCSEA). The animals' development, health, and capacity for food intake were tracked during the whole trial time to ensure their overall well-being.

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Received on 02.08.2022 Modified on 29.08.2022

Research J. Pharm. and Tech 2023; 16(6):2829-2834.

DOI: 10.52711/0974-360X.2023.00466