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Abstract

Despite wide application of sodium nitrite (SN) as food additive, it exhibits considerable side effects on various body organs at high dose or chronic exposure. The aim of this study was to test whether Glycyrrhizic acid (GA) could ameliorate SN-induced toxicity in lung and submandibular salivary gland (SMG). A sample size of 30 adult male albino rats was randomly allocated into 3 groups. Group 1 served as control group. Rats were treated orally with 80 mg/kg of SN in group 2 or SN preceded by (15 mg/kg) GA in group 3. Lung & SMG tissues were used for oxidative stress assessment, examination of histopathological changes, fibrosis (MTC, TGF-β and α-SMA) and inflammation (TNF-α, IL-1β and CD-68). Concurrent administration of GA ameliorated pulmonary and salivary SN-induced toxicity via restoring the antioxidant defense mechanisms with reduction of MDA levels. GA reduced the key regulators of fibrosis TGF-β and α-SMA and collagen deposition. In addition to reduction of inflammatory cytokine (TNF-α, IL-1β) and macrophages recruitments, GA amended both pulmonary and salivary morphological changes. The present study proposed GA as a promising natural herb with antioxidant, anti-inflammatory and antifibrotic effects against pulmonary and salivary SN-induced toxicity.

Keywords

Glycyrrhizic acid sodium nitrite oxidative stress fibrosis lung submandibular gland

Introduction

Sodium nitrite (SN) is an inorganic salt widely used in food industry to give processed meat their characteristic flavor and color, to prevent their spoilage and to delay oxidative rancidity.¹ Apart from the benefits offered by SN, it was reported to exhibit organ toxicity in animals and human at high dose or chronic exposure.^2,3 SN reacts with hemoglobin to produce methaemoglobin with compromised oxygen carrying capacity. Consequently, acute exposure to SN in fatal methemoglobinemia.⁴ Single acute exposure to SN resulted in intestinal damage in rats.¹ On chronic exposure, SN is accompanied by mutagenicity, carcinogenicity, endocrine disturbance, nervous system damage and respiratory dysfunction. By reaction with diet’s amines and amides, SN is converted to nitrosamines and nitrosamides, respectively which trigger oxidative stress, inflammation, and tumorigenesis.⁵ Furthermore, these harmful chemical products can be formed from SN under acidic medium in stomach or under high temperature during charring or overcooking meat. In vitro⁶ and in vivo⁷ studies reported production of highly carcinogenic N-nitrosocompounds from reaction of nitrite with secondary amines and N-alkyl amides at low pH. Currently, exposure to and/or consumption of SN beyond the non-toxic recommended daily intake is alarmingly increasing, especially in high-risk categories, as infants and children. This is attributed to industrial growth, improper agricultural practices, and uncontrolled consumption of processed food.⁸ Therefore, development of therapeutic strategies to halt SN intoxication is of great demand.

Recently, therapeutic application of natural dietary compounds to forestall unfavorable signaling mechanisms triggered by toxins in various body organs is widely used trend in clinical medicine. Glycyrrhizic acid (GA) is the chief constituent of licorice root Glycyrrhiza glabra. It is a triterpenoid saponin traditionally used for treatment of jaundice, bronchitis, and gastritis. Previous studies reported multiple beneficial pharmacological effects for GA including anti-inflammatory, immunity boosting, anti-tumor and anti-fibrotic activities, anti-cariogenic and even antiviral against SARS−Coronavirus.^9
–11 We previously reported protective effect of thymoquinone against SN-induced nephrotoxicity.³ Among various organs adversely affected by SN, Kadry and Ali¹² and Al-Rasheed et al.¹³ reported severe pulmonary injury in SN intoxicated rats. However, whether SN possesses toxic effect on submandibular salivary gland (SMG) has not previously reported.

Intoxicated SMG would directly affect general wellbeing as result of xerostomia, possible rampant caries and decreased immunity of oral cavity. Recently, an expanding prospective of pulmonary salivary gland-type tumors as well as metastasis of certain pulmonary carcinoma into SMG has been linking SMG to precancerous and neoplastic changes in lungs.^14,15 Moreover, both lung and SMG are lately proposed responsible for transmission and replication of Andes Hantavirus (ANDV) that caused 40% fatality rate in human.¹⁶ Herein, we aimed to evaluate the role of oxidative stress, inflammation, and fibrosis in the toxic effect of SN on lung and SMG and to highlight the potential protective role of GA, as an attempt to ameliorate the side effects of inevitable SN exposure on such important organs.

Material and methods

Experimental design

Thirty male Sprague Dawley rats weighing 100–150 g were housed in specific plastic cages with wire mesh lids. All rats were pre-acclimatized to laboratory conditions for one-week pre-experimentation. Rats were kept in regular light cycle of 12/12 h, with 50% relative humidity and free access to food and water. All study procedures were approved by ethical committee of Faculty of Medicine Mansoura University (No: R.20.07.938) and complied to the ARRIVE guidelines of Animal Research. All rats were randomly and equally allocated using the Research Randomizer software (https://www.randomizer.org/) into 3 groups. (1) Control group received phosphate buffer solution. (2) Sodium nitrite-treated (SN) group, rats were treated with (80 mg/kg) SN dissolved in distilled water (Sigma Aldrich, St Louis, MO, USA) (3, 8). (3) Glycyrrhizic acid (GA) treated group, rats were given 15 mg/kg GA, dissolved in distilled water (Sigma-Aldrich, St Louis, MO, USA) followed by SN (80 mg/kg). The selected dose and the experimental procedure applied were based on previous studies. The dose is a rodent equivalent to human’s dose.^17
–19 All treatment modalities were given orally and daily for 3 months.

Euthanasia and sample collection

All rats were anesthetized then euthanized by overdose of halothane.²⁰ Both SMG and lung specimens were collected and processed for further analysis. The sublingual gland was separated from submandibular-sublingual gland complex before processing.

Determination of levels of malondialdehyde (MDA), glutathione (GSH) and total antioxidant capacity (TAC) in lung and SMG tissue

The left halves of lung and SMG were washed with ice cold phosphate-buffered saline (pH = 7.4), then butylated hydroxytoluene and proteolysis inhibitor were added to prevent the proteolysis and oxidation of the samples. After homogenization and centrifugation, total protein content in the supernatants was measured. To evaluate endogenous intoxication of lung and SMG tissues, levels of malondialdehyde (MDA) as a pro-oxidant parameter was determined with the MDA Assay Kit (Biodiagnostic Company, Cairo, Egypt) according to manufacturer’s instructions. Basically, following the addition of thiobarbituric acid, absorbance of formed chromophore was measured at 532 nm. Reduced glutathione (GSH), a cysteine carrier, detoxifies lipid peroxidation products and carcinogens. As a major intracellular antioxidant, levels of GSH were measured (Biodiagnostic Company, Cairo, Egypt) spectrophotometrically to detect a yellow anion that resulted from reduction of 5,5/-dithiobis-(2 nitrobenzoic acid) (DTNB) by GSH at 412 nm.³ Further, to assess the oxidative stress intensity of lung and SMG against SN induced toxicity, TAC (Biodiagnostic Company, Cairo, Egypt) was assayed reflecting the efficiency of enzymatic and non-enzymatic antioxidant defense mechanisms.

Measurement of pro-inflammatory and fibrotic markers in lung and SMG tissue

Based on enzyme-linked immuno-sorbent assay (ELISA), the levels of biochemical parameters in both lung and SMG homogenates were measured using rat transforming growth factor-beta (TGF-β), Interleukin 1-beta (IL-1β) and Tumor necrosis factor-alpha (TNF-α) specific ELISA kits (eBioscience, Inc., San Diego, CA, USA) according manufacturer’s instructions.

Histological, special and immunohistochemical (IHC) stains

The right halves of specimens were fixed, processed into 5 µm paraffin sections, and stained with Hematoxylin and Eosin (H&E),²¹ Masson’s Trichrome (MTC) stain to detect collagen fibers, and Periodic Acid Schiff (PAS) to evaluate the presence and alteration of polysaccharides in the acinar cells, basement membrane and goblet cells.²² For immunostaining, the sections were deparaffinized, blocked with 5% serum for 30 minutes at 20°C and processed for antigen retrieval by heat mediation in a citrate buffer at 20°C for 45 minutes. Then, sections were incubated for 24 hours with primary CD-68 antibody, (ab125212, 1:100) or α-smooth muscle actin (CBL171, Millipore, USA; 1:100) using appropriate secondary antibodies. Negative controls were stained by substituting the primary antibodies by phosphate buffered salin.²³

Evaluation and scoring of special & IHC staining

Assessment was performed by double-blinded calibrated examiners to measure the number of positively stained CD68 cells and to threshold the percentage of positive immunoreactive area of α-SMA and MTC to the total area of each section. An intraclass correlation coefficient was conducted to assess reliability of scores (supplementary file). The score was calculated as follow: 0 → 100% negative expression, and 1 → (>0%–<10%) very mild expression, 2 → (>10%–<30%) mild expression, 3→ (>30%–<50%) moderate and 4 → (>50%) intense positive expression.

Statistical analysis

Statistical package for social sciences (SPSS) (Version 25; Inc, Chicago, IL, USA) was used. Qualitative data was tested for normality using Shapiro-Wilk test. Parametric data were tested by One Way ANOVA (analysis of variance) followed by Tuckey post-hoc test for multiple comparisons. Kruskal–Wallis test followed by post-hoc Dunn’s were used for comparing non-parametric data. P value <0.05 was considered statistically significant. The statistical tests were based on a type 1 error value of 5% (α = 0.05) and on a power of 0.85 sample size, using G*Power 3.1.9.4 program.

Results

Effect of GA administration on SN-induced oxidative stress

Pulmonary homogenates from SN group showed a significant higher MDA level (P = 0.003), a significant lower GSH (P < 0.001) and TAC levels (P < 0.001) compared to control group. GA administration significantly decreased MDA levels (p = 0.006) and restored GSH (P = 0.0016) and TAC levels (P = 0.0004) compared to SN group. Salivary intoxicated SN samples showed significantly higher MDA levels (P < 0.001) and diminished GSH and TAC (P < 0.01, P < 0.001) compared to control group. GA treatment restored levels of both GSH, and TAC compared to SN group (P = 0.0024, 0.0032) respectively and reduced MDA levels compared with SN treated samples (P < 0.001) (Table 1).

Table 1.

Effect of GA administration on SN-induced oxidative stress (malondialdehyde (MDA), reduced glutathione GSH, total antioxidant capacity (TAC) in lung and salivary gland. The data are expressed as mean ± SD, (n = 10). **p < 0.01, ***p < 0.001 compared to normal control, ^##p < 0.01, ^###p < 0.001 compared to sodium nitrite-treated (SN) group.

	Lung			Salivary gland
	Control	SN	GA	Control	SN	GA
MDA (nmol/gm tissue)	42.41 ± 7.91	74.88 ± 6.16**	45.68 ± 3.30^###	27.84 ± 0.56	56.85 ± 0.55***	37.79 ± 1.62^###
GSH (mmol/mg tissue)	5.52 ± 0.82	1.78 ± 0.41***	3.84 ± 0.20^##	4.06 ± .047	1.64 ± 0.35**	4.01 ± 0.48^##
TAC (mmol/100 g tissue)	0.85 ± 0.02	0.48 ± 0.04***	0.70 ± 0.02^###	0.96 ± 0.02	0.73 ± 0.03***	0.91 ± 0.05^##

Effect of GA administration on SN-induced inflammation

SN treatment markedly increased TNF-α and IL-1β levels in both lung and SMG homogenates compared to control group (p < 0.001). However, GA administration suppressed SN-induced TNF-α and IL-1β up-regulation in both pulmonary (p = 0.032, p < 0.001) and SMG (p = 0.011, p < 0.001) homogenates, respectively (Table 2).

Table 2.

Effect of GA administration on SN-induced inflammatory and fibrotic cytokines (tumor necrosis factor-α (TNF-α), tumor growth factor-β (TGB-β), interleukin-1β (IL-1 β). The data are expressed as median and interquartile range (IQR) for TNF-α and TGF-β and as mean ± SD for IL-1β, (n = 10). ***p < 0.001 compared to normal control, ^###p < 0.001 compared to sodium nitrite- treated (SN) group.

	Lung			Salivary gland
	Control	SN	GA	Control	SN	GA
TNF (pg/100 mg tissue)	25.50 (20.70–27.80)	50.40*** (44.00–67.50)	35.20^### (30.60–37.00)	23.00 (20.50–27.80)	52.00*** (50.40–69.70)	37.00^### (30.60–40.20)
TGF (pg/100 mg tissue)	330.00 (300.00–400.00)	855.50*** (800.50–900.00)	500.00^### (450.00–523.50)	176.77 (160.50–220.70)	744.44*** (650.44–800.00)	280.00^### (200.00–360.00)
IL-1 beta (pg/100 mg tissue)	86.17 ± 3.56	460.60 ± 4.04***	260.67 ± 5.09^###	67.33 ± 1.14	235.17 ± 16.37***	154.10 ± 3.98^###

Further, testing CD68 as a marker of macrophage infiltration showed increased bright brown positive IHC staining, reflecting macrophage infiltration into peribronchial, perivascular and interstitial tissue in pulmonary sections from SN treated group. Nevertheless, macrophages were markedly decreased in pulmonary sections from GA group (Table 3, Figure 1A). Similarly, SMG sections from SN group showed positive reaction especially around the highly vacuolated ducts and morphologically distorted acini, whereas GA treated group showed less brown stained cells (Table 3, Figure 1B).

Table 3.

Effect of GA administration on % area of MTC stain, α-smooth muscle actin (α-SMA) and CD86 scoring. Data are expressed as mean ± SD for MTC stain and α-SMA. CD86 scoring data are expressed as median and interquartile range (IQR). *p < 0.05, ***p < 0.001 compared to normal control, ^#p < 0.05, ^###p < 0.001 compared to sodium nitrite-treated (SN) group.

	Lung			Salivary gland
	Control	SN	GA	Control	SN	GA
MTC stain	0.21 ± 0.043	7.08 ± 0.56***	2.17 ± 0.359^###	0.2 ± 0.03	7.13 ± 0.632***	2.16 ± 0.412^###
α-SMA	6.08 ± 1.28	12.05 ± 1.71***	6.59 ± 1.12^###	6.3 ± 0.43	11.01 ± .0.9***	9.1 ± 0.872^###
CD68	0.00 (0.00–0.00)	2.00 (1.00–2.00)*	1.00 (0.00–1.00)^#	0.00 (0.00–0.00)	2.00 (1.00–2.00)*	1.00 (0.00–1.00)^#

Figure 1.

Effect of GA administration on SN-induced inflammation. A) Microscopic pictures of CD68 immunostained lung sections showing, negative staining in control group. In contrast, lung of SN group showed increased expression of bright brown positively stained macrophages infiltrating into peribronchial, perivascular and interstitial tissue (arrows). While in GA group the numbers of infiltrating macrophages were markedly decreased (arrows). (X200, bar 50). B) Microscopic picture of CD68 immunostained SMG sections. Control group showed very minimal reaction interstitially (arrow), while the SN group showed positive reaction especially around the highly vacuolated ducts (arrow) and even intra-acinar in the morphologically distorted acini (arrow head), while GA group showed a lesser number of positively stained macrophages (arrow). (X400, bar 25).

Effect of GA administration on SN-induced fibrosis

Our results revealed increased TGF-β levels in lung and SMG homogenate from SN treated group compared to control group (p < 0.001). In contrast, significant decrease in TGF-β levels was observed in pulmonary and SMG homogenate in GA treated group (p = 0.011) compared to SN treated group (Table 2).

Additionally, pulmonary sections from SN group showed increased bright brown α-SMA positive staining in peribronchial, perivascular and interstitial tissue associated with areas of lesion. However, the immunostaining of α-SMA was markedly decreased in GA group compared to SN group (Table 3, Figure 2A). Likewise, normal sections of SMG showed positive α-SMA reaction, where it binds to microfilament bundles in the myoepithelial cells of the acinar cells and intralobular ducts. It represents the degree of myoepithelial cell proliferation, in the form of brown deposits in their cytoplasm. Positive expression of α-SMA increased in SN treated group with increased number of processes around the ducts and acini. GA group showed lesser degree of α-SMA reaction (Table 3, Figure 2B).

Figure 2.

Effect of GA administration on SN-induced fibrosis. A) Microscopic pictures of α-SMA immunostained lung sections showing normal bright brown positive staining in wall of blood vessels, bronchi, and bronchioles (blue arrows) in Control group. In contrast, SN group lung sections showed increased expression of α-SMA in peribronchial, perivascular and interstitial tissue (yellow arrows) associated with areas of lesion. Amount of α-SMA positive staining in peribronchial, perivascular and interstitial tissue (yellow arrows) was markedly decreased in GA group. (X100, bar 100). B) Microscopic picture of SMG sections stained with α-SMA showing normal brown staining of myoepithelial cells mainly around the acini and interlobular the ducts in Control group. In contrast there was an increase in positive expression of α-SMA with increased number of processes around the ducts (arrow) and acini (arrow heads) in SN group. While there was a decreased α-SMA staining (arrow) in GA group. (X400, bar 25).

Histopathological changes

The control group of both lung (Figure 3A) and SMG (Figure 3B) showed almost normal morphology indicating proper experimental conditions. In SN group, pulmonary sections showed peribronchial lymphoid hyperplasia and degeneration in alveolar epithelium, while in SMG, the duct system seemed to be more affected by SN toxicity than the acini. Loss of acinar structures, granular convoluted duct changes were also observed. Moreover, widening and thickening of interstitial spaces and tissues were marked in both lung and SMG sections. Thickening and fibrosis were much confirmed with the MTC stain results in both lung (Table 3, Figure 4A) and SMG sections (Table 3, Figure 4B) from SN group. Fibrous deposition in both lung (Table 3, Figure 4A ) and SMG (Table 3, Figure 4B) sections was minimal with reduction of congestion and thickening of interstitial in both tissues in GA treated group.

Figure 3.

A) Microscopic pictures of H&E stained lung sections showing normal alveoli, bronchioles, interstitial tissue, and blood vessels in Control group. In contrast, lung of SN group showed perivascular leukocytic cells aggregation (black arrows), peribronchial lymphoid hyperplasia (green arrow) and degeneration in alveolar epithelium (blue arrows). Marked improvements were noticed in GA group including mild thickening of interstitial tissue (yellow arrows), mild congestion and few perivascular leukocytic cells aggregation. (X100, bar 100 and X400, bar 50). B) Microscopic picture of H&E stained SMG sections. Normal group showed acini with narrow lumen bordering pyramidal cells with and basal nuclei (arrows), interlobular ducts with tall columnar cells and basal nuclei (arrow heads). SN group showed loss of normal duct structures (arrows), thickening of interlobular duct wall with collapse of the lumen (curved arrows) with hyalinized homogenous areas (arrow heads), nuclear changes (n) in both duct and acini and congested blood vessels (*). SN group also showed loss of parenchymal tissue and wide interstitial spaces (arrows). While GA group revealed considerable restoration of normal glandular architecture and regeneration of parenchymal including GCD and connective tissue components. Some ductal (arrows) and nuclear (n) changes were also observed. GCT; granular convoluted tubules, A; acini. (X400, bar 25).

Figure 4.

A) Microscopic pictures of MTC stained lung sections showing no fibrous tissue deposition in interstitial tissue in Control group. In contrast, lung of SN group showed blue stained fibrous tissue deposition peribronchial, perivascular and in interstitial tissue associated with areas of lesion (arrows). Fibrous deposition was minimal to absent peribronchial, perivascular and in interstitial tissue (arrows) in GA group. (X100, bar 100 and X400, bar 50). B) Microscopic picture of rat SMG stained with MTC showing negative staining except for some fibers surrounding the ducts in Control group. In contrast there was a thickened periductal fibrous connective tissue (arrow) and fibrous thickening of the wall of congested blood vessels (arrow heads) in addition to some interacinar fibrous deposition SN group, while there was a decrease in amount and thickness of fibrous tissues (arrow) in GA group. (X100, bar 100 and X400, bar 25).

PAS stain revealed normal lung structure, continuous epithelial lining of bronchioles with no goblet cells in normal control group. In contrast, marked increase in number of goblet cells in epithelial lining of bronchioles with deposition of pink stained mucus admixed with leukocytes in their lumens were observed in SN treated group. Goblet cells were markedly decreased to absent in bronchial epithelial lining of GA treated group (Figure 5A). In addition, PAS stained SMG sections showed continuous basement membrane surrounding acini and the secretory granules in acinar cells in control group. SN treated group showed loss of continuity of epithelial lining surrounding duct system, while GA treated group almost regained normal basement membrane (Figure 5B).

Figure 5.

A) Microscopic pictures of PAS stained lung sections showing normal epithelial lining of bronchioles with no goblet cells (blue arrows) in Control group. In contrast, lung of SN group showed marked increase in number of goblet cells in epithelial lining of bronchioles (yellow arrows) with deposition of pink stained mucus admixed with leukocytes in their lumens (black arrows). Goblet cells markedly decreased to absent in epithelial lining of bronchioles (yellow arrows) in GA group. (X100, bar 100 and X400, bar 50). B) Microscopic picture of PAS stained SMG sections showing positive staining of basement membrane (BM) delaminating the acini and ducts (arrow) in Control group (arrow) while discontinuities of the epithelial lining of interlobular ducts (arrow) were observed in SN group. GA group showed almost restoration and less destructed BM (arrow). (X400, bar 25).

Discussion

Despite the importance of food additives for maintenance and/or improvement of food quality, current consumption of great amounts of these additives with reported predisposition to their toxic hazards has prompted the scientific community to oversee this issue. SN is widely used in food technology. Moreover, increased exposure to high levels of SN in contaminated drinking water has been reported.²⁴ This inevitable vulnerability to SN toxicity raises the importance of delineating its possible harmful effects on various body organs as well as detection of appropriate protective strategies.²⁵ In the present study, we reported SN-induced toxicity on lung and SMG with protective effect of concomitant GA administration. This inference was based on the findings that GA treatment combated SN-induced oxidative stress, inflammation, and fibrosis.

Multiple mechanisms have been proposed for SN induced toxicity, among them oxidative stress is a crucial player. It was reported that administration of SN was accompanied by disruption of redox homeostasis in various body organs. This was manifested as increased free radical production, lipid peroxidation and reduced endogenous antioxidant defense species responsible for quenching of produced free radicals. On the other hand, natural antioxidants have been shown to preserve liable organs against SN-induced oxidant/antioxidant imbalance.^3,8,26 In this context, our results demonstrated increased MDA levels in lung and SMG concomitant with compromised antioxidant capacity in SN treated rats. Whereas GA administration improved redox status, attenuated lipid peroxidation, and restored total antioxidant capacity both in lung and salivary gland. Consistent with our finding, GA has been shown to be neuroprotective²⁷ and cardioprotective²⁸ via antioxidant mechanism. Moreover, Zhao et al.²⁹ demonstrated that GA attenuated oxidative stress in a model of sepsis-induced acute lung injury.

Stimulation of inflammatory response featured by recruitment of macrophages concomitant with up-regulated production of inflammatory cytokines was outlined as a cardinal mechanism in SN-induced organ injury.^3,30
–32 In line, we found increased macrophage infiltration accompanied by increased TNF-α and IL-1β levels in lung and SMG of SN intoxicated rats. In addition, PAS staining demonstrated increased goblet cells, mucus, and leukocytes in epithelial lining of bronchioles and discontinuity of epithelial lining of SMG ducts. However, GA administration reversed inflammatory status triggered by SN. Our results were in agreement with previous finding that reported anti-inflammatory capacity of GA in various inflammatory conditions^33,34 including lung inflammation both in vivo³⁵ and in vitro.³⁶

Persistent uncontrolled inflammation leads ultimately to scar tissue formation and fibrosis. Inflammatory cytokines produced by recruited inflammatory cells, mainly TNF-α and IL-1β, activate TGF-β a crucial player in over synthesis and accumulation of extracellular matrix proteins during fibrosis, resulting in tissue repair. Many reports revealed that TGF-β mediates lung and SMG fibrosis via activation of fibroblasts, induction of α-SMA and enhancement of ECM synthesis. Therefore, TGF-β represents a central fibrotic protein that can be targeted for inhibition of fibrotic events in both lung and SMG fibrosis.^37,38 SN toxicity is accompanied by stimulation of TGF- β signaling with consequent triggering of the fibrotic processes in various organs including lung.¹³ In agreement, our data indicated increased expression of TGF-β, α-SMA and collagen in pulmonary and salivary gland tissue of SN treated rats. This fibrotic condition was markedly relieved by GA administration. Similarly, GA anti-fibrotic capability was previously demonstrated in experimental models of liver³⁹ and lung⁴⁰ fibrosis.

Conclusion

In conclusion, our study presented protective capacity of GA against pulmonary and salivary gland toxicity induced by SN in rats. This was mediated via multiple mechanisms including restoration of oxidant/antioxidant balance, mitigation of inflammatory status and suppression of fibrosis in rat lung and salivary gland (Figure 6).

Figure 6.

Graphical abstract.

Supplemental material

Supplemental Material, Supplementary-data-1 - Glycyrrhizic acid ameliorates sodium nitrite-induced lung and salivary gland toxicity: Impact on oxidative stress, inflammation and fibrosis

Supplemental Material, Supplementary-data-1 for Glycyrrhizic acid ameliorates sodium nitrite-induced lung and salivary gland toxicity: Impact on oxidative stress, inflammation and fibrosis by Amira M Elsherbini, Nadia M Maysarah, Mohamed El-Sherbiny, Mohammed MH Al-Gayyar and Nehal M Elsherbiny in Human & Experimental Toxicology

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Nehal M Elsherbiny

Supplemental material

Supplemental material for this article is available online.

References

Ansari

Ali

Arif

, et al. Acute oral dose of sodium nitrite induces redox imbalance, DNA damage, metabolic and histological changes in rat intestine. PloS One 2017; 12(4): e0175196.

Adewale

Samuel

Manubolu

, et al. Curcumin protects sodium nitrite-induced hepatotoxicity in Wistar rats. Toxicol Rep 2019; 6: 1006–1011.

Elsherbiny

Maysarah

El-Sherbiny

, et al. Renal protective effects of thymoquinone against sodium nitrite-induced chronic toxicity in rats: impact on inflammation and apoptosis. Life Sci 2017; 180: 1–8.

Chui

JSW

Poon

Chan

, et al. Nitrite-induced methaemoglobinaemia—aetiology, diagnosis and treatment. Anaesthesia 2005; 60(5): 496–500.

Cvetković

Živković

Lukić

, et al. Sodium nitrite food poisoning in one family. Forensic Sci Med Pathol 2019; 15(1): 102–105.

Aoyagi

Matsukura

Uchida

, et al. Induction of liver tumors in Wistar rats by sodium nitrite given in pellet diet. J Natl Cancer Inst 1980; 65(2): 411–414.

Hecht

. Approaches to cancer prevention based on an understanding of N-nitrosamine carcinogenesis. Proc Soc Exp Biol Med 1997; 216(2): 181–191.

Hamdan

Al-Gayyar

Shams

MEE

, et al. Thymoquinone therapy remediates elevated brain tissue inflammatory mediators induced by chronic administration of food preservatives. Sci Rep 2019; 9(1): 7026.

El-Saber Batiha

Magdy Beshbishy

El-Mleeh

, et al. Traditional uses, bioactive chemical constituents, and pharmacological and toxicological activities of Glycyrrhiza glabra L. (Fabaceae). Biomolecules 2020; 10(3): 352.

10.

Hoever

Baltina

Michaelis

, et al. Antiviral activity of glycyrrhizic acid derivatives against SARS−coronavirus. J Med Chem 2005; 48(4): 1256–1259.

11.

Sidhu

Shankargouda

Rath

, et al. Therapeutic benefits of liquorice in dentistry. J Ayurveda Integr Med. 2018; 11(1): 82–88.

12.

Kadry

Ali

. Downregulation of HIF1-α, Smad-2, AKT, and Bax gene expression in sodium nitrite-induced lung injury via some antioxidants. J Biochem Mol Toxicol 2017; 31(7): e21909.

13.

Al-Rasheed

Fadda

Attia

, et al. Quercetin inhibits sodium nitrite-induced inflammation and apoptosis in different rats organs by suppressing Bax, HIF1-α, TGF-β, Smad-2, and AKT pathways. J Biochem Mol Toxicol 2017; 31(5): e21883.

14.

Falk

Weissferdt

Kalhor

, et al. Primary pulmonary salivary gland-type tumors: a review and update. Adv Anato Pathol 2016; 23(1): 13–23.

15.

Lee

Kim

Yoon

, et al. Metastatic small cell neuroendocrine carcinoma of the submandibular gland from the lung: a case report. Medicine (Baltimore) 2020; 99(4): e19018.

16.

Pizarro

Navarrete

Mendez

, et al. Immunocytochemical and ultrastructural evidence supporting that Andes hantavirus (ANDV) is transmitted person-to-person through the respiratory and/or salivary pathways. Front Microbiol 2020; 10: 2992.

17.

Khan

Lateef

, et al. Glycyrrhizic acid suppresses the development of precancerous lesions via regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of Wistar rats. PloS One 2013; 8(2): e56020.

18.

Nair

Jacob

. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016; 7(2): 27–31.

19.

Reagan-Shaw

Nihal

Ahmad

. Dose translation from animal to human studies revisited. FASEB J 2008; 22(3): 659–661.

20.

Leary

SLUW

Anthony

Cartner

, et al. AVMA guidelines for the euthanasia of animals. Schaumburg, IL: American Veterinary Medical Association, 2013.

21.

Feldman

Wolfe

. Tissue processing and hematoxylin and eosin staining. Methods Mol Biol (Clifton, NJ) 2014; 1180: 31–43.

22.

El Sadik

Mohamed

El Zainy

Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: biochemical, histological and ultrastructural study. PloS One 2018; 13(10): e0205372.

23.

Yamashita

Katsumata

. Heat-induced antigen retrieval in immunohistochemistry: mechanisms and applications. Methods Mol Biol (Clifton, NJ) 2017; 1560: 147–161.

24.

Ansari

Mahmood

. Carnosine and N-acetyl cysteine protect against sodium nitrite-induced oxidative stress in rat blood. Cell Biol Int 2018; 42(3): 281–293.

25.

Hassan

El-Agmy

Gaur

, et al. In vivo evidence of hepato- and reno-protective effect of garlic oil against sodium nitrite-induced oxidative stress. Int J Biol Sci 2009; 5(3): 249–255.

26.

Ansari

Khan

Mahmood

. Protective effect of carnosine and N-acetylcysteine against sodium nitrite-induced oxidative stress and DNA damage in rat intestine. Environ Sci Pollut Res Int 2018; 25(20): 19380–19392.

27.

Ojha

Javed

Azimullah

, et al. Glycyrrhizic acid attenuates neuroinflammation and oxidative stress in rotenone model of Parkinson’s disease. Neurotox Res 2016; 29(2): 275–287.

28.

Liang

Sun

, et al. Glycyrrhizic acid ameliorates myocardial ischemic injury by the regulation of inflammation and oxidative state. Drug Des Devel Ther 2018; 12: 1311–1319.

29.

Zhao

Wang

, et al. Glycyrrhizic acid prevents sepsis-induced acute lung injury and mortality in rats. J Histochem Cytochem 2016; 64(2): 125–137.

30.

Attia

Fadda

Al-Rasheed

, et al. Carnosine and l-arginine attenuate the downregulation of brain monoamines and gamma aminobutyric acid; reverse apoptosis and upregulate the expression of angiogenic factors in a model of hemic hypoxia in rats. Naunyn Schmiedebergs Arch Pharmacol 2020; 393(3): 381–394.

31.

Alyoussef

Al-Gayyar

MMH

. Thymoquinone ameliorates testicular tissue inflammation induced by chronic administration of oral sodium nitrite. Andrologia 2016; 48(5): 501–508.

32.

Sherif

Al-Gayyar

. Cod liver oil in sodium nitrite induced hepatic injury: Does it have a potential protective effect? Redox Rep 2015; 20(1): 11–16.

33.

Kao

T-C

Shyu

M-H

Yen

G-C

. Glycyrrhizic acid and 18β-glycyrrhetinic acid inhibit inflammation via PI3K/Akt/GSK3β signaling and glucocorticoid receptor activation. J Agric Food Chem 2010; 58(15): 8623–8629.

34.

Luo

Jin

Kim

I-D

, et al. Glycyrrhizin attenuates kainic acid-induced neuronal cell death in the mouse hippocampus. Exp Neurobiol 2013; 22(2): 107–115.

35.

Menegazzi

Di Paola

Mazzon

, et al. Glycyrrhizin attenuates the development of carrageenan-induced lung injury in mice. Pharmacol Res 2008; 58(1): 22–31.

36.

Matsui

Matsumoto

Sonoda

, et al. Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line. Int Immunopharmacol 2004; 4(13): 1633–1644.

37.

Arribillaga

Dotor

Basagoiti

, et al. Therapeutic effect of a peptide inhibitor of TGF-β on pulmonary fibrosis. Cytokine 2011; 53(3): 327–333.

38.

Woods

Camden

El-Sayed

, et al. Increased expression of TGF-β signaling components in a mouse model of fibrosis induced by submandibular gland duct ligation. PloS One 2015; 10(5): e0123641.

39.

Liang

Guo

X-L

Jin

, et al. Glycyrrhizic acid inhibits apoptosis and fibrosis in carbon-tetrachloride-induced rat liver injury. World J Gastroenterol 2015; 21(17): 5271–5280.

40.

Gao

Tang

, et al. Glycyrrhizic acid alleviates bleomycin-induced pulmonary fibrosis in rats. Front Pharmacol 2015; 6: 215.

Supplementary Material

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Glycyrrhizic acid ameliorates sodium nitrite-induced lung and salivary gland toxicity: Impact on oxidative stress,inflammation and fibrosis

Abstract

Keywords

Introduction

Material and methods

Experimental design

Euthanasia and sample collection

Determination of levels of malondialdehyde (MDA), glutathione (GSH) and total antioxidant capacity (TAC) in lung and SMG tissue

Measurement of pro-inflammatory and fibrotic markers in lung and SMG tissue

Histological, special and immunohistochemical (IHC) stains

Evaluation and scoring of special & IHC staining

Statistical analysis

Results

Effect of GA administration on SN-induced oxidative stress

Effect of GA administration on SN-induced inflammation

Effect of GA administration on SN-induced fibrosis

Histopathological changes

Discussion

Conclusion

Supplemental material

Supplemental Material, Supplementary-data-1 - Glycyrrhizic acid ameliorates sodium nitrite-induced lung and salivary gland toxicity: Impact on oxidative stress, inflammation and fibrosis

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

Supplemental material

References

Supplementary Material