Anti-inflammatory effect of Phytomarine R-L compound against lipopolysaccharide- induced pulmonary injury in BALB/c mice

FFHD Page 666 of 679 Functional Foods in Health and Disease 2022; 12(11):666-679 Research Article Open Access Anti-inflammatory effect of Phytomarine R-L compound against lipopolysaccharideinduced pulmonary injury in BALB/c mice Janani Gopi1, Francesco Marotta2*, Sushmitha Sriramulu1, Antara Banerjee1, N.R. Rajesh Kanna3, Vignesh Palani4, Antonio Ayala5, Fang He6, R. Vijayashree3, Surajit Pathak1* 1Department of medical biotechnology, Faculty of Allied Health Sciences, Chettinad Academy of Research, and Education, Chettinad Hospital and Research Institute (CHRI), Chennai , India; 2ReGenera R&D International for Aging Intervention and Vitality and Longevity Medical Science Commission, FEMTEC World Federation, Milan, Italy; 3Department of Pathology, Faculty of Medicine, Chettinad Academy of Research and Education, Kelambakkam, Tamil Nadu, India; 4Faculty of Medicine, Chettinad Hospital and Research Institute (CHRI), Chennai, India; 5Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, Spain; 6Department of Nutrition and Food hygiene, West China School of Public Health and West China Fourth Hospital, Sichuan University, Sichuan China. *Corresponding Author: Surajit Pathak, PhD, Professor, Department of medical biotechnology, Faculty of Allied Health Sciences, Chettinad Academy of Research, and Education, Chettinad Hospital and Research Institute (CHRI), Chennai - 603 103, India Submission Date: September 20th, 2022; Acceptance Date: November 14th, 2022; Publication Date: November 22nd, 2022 Please cite this article as: Gopi, J., Marotta F., Sriramulu S., Banerjee A., Kanna N. R., Palani V., Ayala A., He F., Vijayashree R., Pathak S. Anti-inflammatory effect of Phytomarine R-L compound against lipopolysaccharideinduced pulmonary injury in BALB/c mice. Functional Foods in Health and Disease 2022; 12(11):666-679. DOI: https://www.doi.org/10.31989/ffhd.v12i11.1014 ABSTRACT Background: This study deals with the induction of lipopolysaccharide induced inflammation in animal model and treating this condition using phytomarine R-L compound (Bloomin’Age, Science of Living, Milan Italy) that is known to possess the anti-inflammatory properties and eventually determine their characteristics through the in-vivo experimentation. Objective of the study: The present study was subjected to test the therapeutic anti-inflammatory/anti-oxidative and regenerative property of this phytomarine (SBF-LF) in an LPS-induced lung injury in mice models. for four consecutive weeks followed by the oral administration of R-L compound at a concentration of 50 mg/kg body weight intermittently. Broncho-alveolar lavage fluids (BALF) as well as the lung tissues were periodically collected from Methods: Mice received an intra-nasal administration of LPS at a concentration of 1 mg/ml weekly Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 667 of 679 the control and experimental groups for histological examination. Likewise, the lung tissue homogenate obtained from induction and treatment groups was assayed for myeloperoxidase (MPO) assay, superoxide dismutase (SOD) activity and elastase assay. Results: The results illustrated that LPS- induced mice group demonstrated an increased inflammatory cell infiltration causing an acute and persisting neutrophilic accumulation in the lung parenchyma with alveolar congestion. Furthermore, the influx of macrophages, lymphocytes as well as inflammatory mediators was observed during the BALF analysis. The treatment with R-L compound resulted in a significant decline in the permeation of inflammatory cells as observed by lung histology and BALF analysis. Conclusion: Thus, the exploitation of R-L compound with the experimental groups along with other assay methods confirmed its robust anti-inflammatory and antioxidant properties and suggested epithelial regenerative capacity. Further analyzing the efficacy of the R-L compound might help understanding its potential as a therapeutic option in clinical settings. Keywords: Lungs, BALB/c mice, Lipopolysaccharide, anti-inflammatory, R-L compound ©FFC 2022. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License (http://creativecommons.org/licenses/by/4.0) Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 268 of 679 INTRODUCTION Inflammation is an adaptive response implicated in the aging process and underlying a broad spectrum of pathological as well as physiological processes affecting protective cellular mechanisms whenever tissue injury and infections occur. Acute inflammation mainly encompasses the migration of neutrophils from the vasculature into the inflammatory region that results in an interim matrix with the production of chemoattractant factors through platelets at the site of inflammation which inlay for a period of two to six weeks and gradually shifts to stage of chronic inflammation [1]. Our research work predominantly concentrates on the inflammatory condition in lungs causing asthma, chronic obstructive pulmonary disease involving the chronic bronchitis, as well as emphysema, pneumonia, and acute respiratory distress syndrome. Conscription of leukocytes at the site of inflammation mainly involves the functional response of a significant chemotactic action on cytokines [2]. Thus, the delivery of pathogens is known to be a potent method of inspecting the host’s inflammatory reaction [3]. Toxic substances such as LPS, an endotoxin from cell wall of gram-negative bacteria and present in tobacco smoke as well as in a range of environmental and occupational dusts exposure, can ultimately lead to acute and chronic pulmonary inflammation through the infiltration of neutrophils and monocytes into the alveoli. Thus, LPS is a recognized trigger model for investigating the acute inflammatory response. This is associated to the release of pro-inflammatory cytokines encompassing the tumor necrosis factor alpha (TNF-α), interleukin-1β and interleukin-6. LPS-induced inflammation model in mice reiterates the facets of inflammatory cascade that are interconnected with the pulmonary inflammation and lung ailments in humans [4]. Sequentially, LPS provokes the immunological reactions by interrelating with the membrane receptor CD14 which is a 55 kDa glycosyl phosphatidylinositol connected membrane protein that stimulates the production of multiple cytokines while the protein kinases particularly Fgr, Lyn and Hck engage mainly in the signalling pathway of LPS. Within minutes of LPS stimulation, there is a copious proteins efflux which are tyrosine phosphorylated where the MAP kinases p42, p44 and p38 are enzymatically active and trigger the release of macrophages [5]. Concomitantly, the signalling mechanism of Type I integral protein involves the Toll like receptor 4 (TLR-4) engrosses the recruitment of intracellular adaptor proteins that transmit signals from the TIR domain, initiating the protein kinases as well as transcription factors that provoke the release of inflammatory mediators [6] whose involvement is known to be linked with acute and chronic inflammation in human. Therefore, using intranasal delivery for delivering substances in the bronchus mirrors a clinically compatible and interventional methodology [7, 8]. Over the last decades, the prominence of natural compounds such as the plants and marine species naturally containing many active chemicals have been studied for their potentially health benefits, either preventative or therapeutic. To refrain from the utilization of classic medicine, the prominence of functional foods along with the phytomedicine might sustain the ministrations of the prevailing medical conditions. Functional foods are not like dietary supplements and are rather traceable through natural compounds that can be taken through normal diet. The Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 269 of 679 promising features of functional food can be employed against viral infections, heart disease, respiratory disease, inflammation and cancer. Moreover, the plant-based components of functional foods can furnish both taste as well as food aroma that can be employed as expectorant, antitussive, antimicrobial and irritation soothers in pulmonary medicine [9]. For instance, flavonoids display a broad array of pharmacological activities dealing with the modulation as well as progression of several pulmonary disease conditions [10 - 13]. Some, albeit still extremely limited, experimental studies have investigated the therapeutic potential of marine extracts in LPS-induced lung injury [14, 15]. Rhodiola rosea, which is mostly found in Europe and Southeast Asia, has a rich polyphenol content (41.4 ± 3.41%) [16] and its salidroside moieties are known to possess various pharmacological activities, such as antioxidant, anti-inflammation, anti-apoptotic, antidepressant, anti-aging, neuroprotective, cardioprotective effects while alleviating cigarettesmoking experimental COPD [17]. Another interesting compound which attracted our research interested was a lipoprotein fraction obtained without chemicals from T. trachurus fish from the Galician coast of the Atlantic Ocean. Lipofishin, i.e., a subclass under the proteolipids, which forms a new group of lipoproteins extracted from the muscle of Trachurus fish have been demonstrated to possess anti-inflammatory properties [18]. After years of research, in 2021, we devised a phytomarine compound (Bloomin’Age, Science of Living, Milan Italy) by accurately mixing of high-salidroside specific bioactive fractions (SBF) isolated from the Rhodiola rosea and the lipoprotein extracted from the muscle of Trachurus trachurus fish (Rhodiola rosea plant extract and Lipoproteins from Trachurus sp). Our previous results showed the significant anti-inflammatory, antioxidant, potential of R-L compound [19]. Thus, the aim of the present study was to test the therapeutic anti-inflammatory/anti-oxidative and regenerative property of this phytomarine (SBF-LF) in an LPS-induced lung injury in mice model. METHODS Reagents: LPS, phosphate buffered saline (PBS), sodium phosphate buffer, Ethylene diamine tetra-acetic acid (EDTA), Sodium hydroxide (NaOH), Concentrated sulphuric acid (H2SO4), Glycerin, Giemsa stain, ethanol, methanol, saline, DNase free water and R-L compound, 10% formaldehyde, Xylene, Tris, Ethylenedi-aminetetra- acetic acid disodium salt dihydrate (Na2EDTA-2H2O), pyrogallol, ammonium chloride, potassium bicarbonate, 3, 3’, 5, 5’-Tetramethylbenzidine (TMB), dimethyl sulphoxide (DMSO). Rhodiola SBF and LF-Trachurus sp (R-L compound) were a kind gift from ReGenera R&D International for Aging Intervention, Italy. Animals: Healthy female BALB/c mice (Mus musculus) aged 8 to 12 weeks old weighing about 20-25 grams approximately were housed in animal house, Chettinad Hospital and Research Institute (CHRI) with the approval number: IAEC 4/ Proposal 29/ A. Lr: 11/ Dt: 20.12.18. They were placed in plastic cages with adsorbent bedding matter with 12h light/dark cycle under standard conditions. Further, ad-libitum feeding of water and food pellet was provided during the in vivo experimentation. LPS-induced lung inflammation: For induction of acute phase of inflammation in lungs, 1 mg of lipopolysaccharide was dissolved in 1ml of sterile saline. Then, each mouse was administered 10µl of LPS (1 mg/ml dose concentration) intra-nasally (IN) to the nares of the mice through micropipette by lightly anaesthetizing with isoflurane and was given once in every week for four consecutive weeks [20]. Treatment Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 270 of 679 of R-L compound was subjected to oral administration with a specific concentration 50 mg/kg body weight was predetermined for the drug treatment. Thus, the R-L compound was given orally to the BALB/c mice at a specific dose concentration of 15 mg/ml (i.e., each mice received 1.5 mg for approximately ~ 30g body weight) at regular time intervals (1st, 3rd, 5th, 7th day of week 5) according to protocol [21]. Experimental groups: Mice were randomized and divided into three groups. Group 1 (n=6) received oral administration of phosphate buffered saline (PBS) which was allotted as negative control. Group 2 (n=6) received LPS (1 mg/ml) which was administered intra-nasally once a day in a week for 4 consecutive weeks and they were sacrificed at the end of four weeks which was allotted as induction/positive control group. Group 3 (n=6) received a selected oral administration of 50µl of R-L compound from week 5 continuously at regular intervals after LPS induction to evaluate its effect on the biochemical parameters (Fig.1). Figure 1: Experimental Design of the study Tissue Collection and Processing: After instillation of deep anesthesia, the animals were euthanized, and the lungs were collected appropriately from the experimental groups. Mice lungs were obtained for light microscopy without induction or perfusion with fixatives to avoid any translocation of leukocytes within the lung parenchyma or vessels. A portion of lung was used for histology by fixing it in 10% formaldehyde and the remaining portion of lung was stored at -20°C till homogenization and processed for further experimental studies. Histology assessment of lung tissue: Lung tissues were fixed and embedded in paraffin. Tissue sections were cut at 4–5 μm from paraffin embedded lung tissues and mounted on glass slides for hematoxylin and eosin stain and examined by an optical microscope [22]. Analysis of BALF: The experimental protocol of BALF collection involves euthanizing the mouse through inhalation of carbon-di-oxide gas and taking them back to the laminar airflow chamber. Then, mice were moistened with ethanol and positioned in supine Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 271 of 679 position by pinning the legs with needles on Styrofoam boards. Dissection of the mice was performed, the organs were exposed, and the fluid was accurately collected about ten times in an Eppendorf tube and stored in ice. The processing of the BALF involved centrifuging the fluids for 10 minutes at 4  C and resuspending the cell pellet in 200 μl of PBS. This was followed by centrifuging the fluids for 10 minutes at 4  C and resuspending the cell pellet in 100 to 200 μl of ACK lysis buffer and placing it on ice for 10 minutes to lyse the RBCs. 1 ml of PBS was added to stop the lysis. Finally, the fluids were centrifuged again at 800G for 10 minutes at 4  C and the cell pellet was resuspended in PBS and used for the experimental procedures and assay methods [23]. Estimation of BALF by Giemsa stain: Bronchoalveolar lavage fluid was centrifuged at 1500 revolutions per minute for 10 minutes and red blood cells were extracted by hypotonic lysis. After a second centrifugation the pellet was dissolved in 500 µl saline. For differential cell count analysis, the pulmonary fluids were diluted with saline to retrieve 1 x 105 cells/100 µl of saline. Polymorphonuclear cells and macrophages were identified by morphological examination of smears and stained with Wright-Giemsa solution. Elastase assay: The elastase assay was carried out for the lung tissues. Elastase stock has a concentration of 3.33 mg/ml which is to be dissolved in sterile H2O and the substrate (N-succinyl- ala-ala-ala-p-nitroanilide) in 1.6mM of buffer. 50 µl of tissue supernatant were combined with the elastase enzyme followed by incubation for 15 minutes. Then, substrate was added to the above reaction mixture and absorbance was read at 402nm. Myeloperoxidase (MPO) assay: The MPO assay was carried out for the lung tissues. The peroxidase activity with 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) was measured suitably in the spectrophotometer. Lung tissue supernatant was combined with 0.75 mM hydrogen peroxide (H2O2) and TMB solution and the plate was incubated at 37oC for 5 min. The reaction was stopped by adding 2M sulphuric acid (H2SO4) and absorption was measured at 450 nm to estimate MPO activity [24]. Superoxide dismutase (SOD) activity: SOD activity in the cell extract was estimated by measuring its ability to inhibit the auto-oxidation of pyrogallol according to the method of Markland [25]. 1ml of assay mixture containing 0.05M sodium phosphate buffer (pH8.0), 0.01M EDTA and 0.27mM pyrogallol (Solution of pyrogallol was made in100mM HCl). The absorbance was measured for 5min at 420nm.The enzyme activity was expressed as U/mg protein, where1U is the amount of enzyme required to bring about 50% inhibition of the auto-oxidation of pyrogallol. Statistical Analysis: Data was obtained from independent experiments performed in triplicate manner and were presented as mean ± SEM. The unpaired student t test was used to determine significant differences between two groups of data. The p values of <0.05, <0.01 and <0.001 were considered as statistically significant and are indicated by asterisks (*, ** and ***) respectively. All the data was analyzed with Graph Pad Prism 5.0 software. RESULTS Animal disease activity index of the mice was analyzed by interpreting the change in the body weight, input of water, and food consumption. Thus, changes in body Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 272 of 679 mass were assessed during the in vivo experiment. Initially, in LPSfed mice, there was a gradual loss of body weight and water and food intake when compared to normal mice, whereas, in LPS + R-L compound fed mice there was a marginal gain in the body mass, water and food consumption (Fig. 2-a, 2-b and 2-c). During animal experiments, the veterinary pulse oximeter was used to measure the saturated peripheral oxygen (SpO2) level in all groups at regular time intervals during LPS induction as well as under R-L compound treatment. From graphical representation, Fig. 2-d depicts a gradual SpO2 decline in the LPS induced mice in contrast to control mice, whereas a slight increment in the LPS + R-L compound treatment to the lung injury mice model. Figure 2: Effect of body mass changes in mice (2-a) was observed during the LPS induction as well as during R-L treatment; (2-b) Water and food consumption was observed during LPS induction; (2-c) Water and food consumption was monitored during R-L treatment; Pulse oximeter reading (2-d) during LPS induction and R-L treatment. Histology assessment of lung tissues: Control mice showed the normal histological structure of the lung sections stained with hematoxylin and eosin. Microscopically, the control tissue section was seen with its lung parenchyma exposed to normoxic condition with alveolar septa and pulmonary capillaries which are found to be preserved (Fig. 3-a). In LPS-induced mice group, the lung tissue sections showed alveolar congestion and enlargement, hemorrhage, infiltration, or aggregation of inflammatory cells such as macrophages, neutrophils and even lymphocytes in airspaces, engorgement of the blood vessel walls and Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 273 of 679 thickening of the alveolar walls as the significant signs of the progression of the disease condition (Fig. 3-b). Thus, the treatment of inflammatory lung changes with the drug (R-L compound) were found to impede the inflammatory cell infiltration in the lung parenchyma as well as in the alveolar and bronchial walls suggesting the recovery of lung tissue to the normoxic condition (Fig. 3- c). Figure 3: Effect of R-L on lung inflammation. Lung sections were obtained from control mice (a) group; LPS treated mice (b) group, LPS + R-L compound mice group (c). After hematoxylin and eosin staining, histological examination of lung tissue sections was observed under light microscopy at 20X magnifications. BALF smear stained using Giemsa stain: The processed BALF was smeared in a glass slide and fixed with methanol followed by Giemsa stain. The smeared glass slide was observed with a compound microscope at 10X magnification. Therefore, in LPS treated group, the BALF cytological analysis revealed that three distinct types of alveolitis were observed namely the lymphocytes, neutrophils, and eosinophils. Especially, in Fig. 4-b, the presence of alveolar casts of amphophilic, amorphous material in honeycomb pattern of cells were observed, the infiltration of inflammatory cells in BALF and in the lung, parenchyma was observed following exposure to LPS in mice. Alveolar macrophages are found which is associated with the acute phase of inflammation respectively. Then, in Fig. 4-c the R-L compound treated group was observed with a minimal level of inflammatory cells accumulation in comparison with LPS treated group. Figure 4: Microscopic images of bronchoalveolar lavage fluid (BALF) stained with Giemsa. Control group 4(a), LPS treated mice group 4(b); LPS + R-L compound treatment group 4(c) were observed at 10X magnification. BAL cells depict eosinophils, neutrophils, macrophages, and lymphocytes in LPS challenged mice. Functional Foods in Health and Disease 2022; 12(4):666-679 FFHD Page 274 of 679 Colorimetric Assay Methods I. Elastase Assay: The enzymatic activities of elastase in control and treatment groups are summarized in Fig. 5- a. The elastase activity significantly increased in the LPS treated group because the alveoli lost their elasticity and secreted the enzymes which are known to be at higher levels in comparison with LPS + R-L treated mice group which exhibit a substantial decrease in their enzymatic reaction. II. Myeloperoxidase Assay: In myeloperoxidase assay, the absorbance is inversely proportional to the level of MPO enzyme. The enzymatic activities of myeloperoxidase in control and treatment groups are summarized in Fig. 5-b. The tissue lysate depicts an increased level of MPO activity in the LPS treated mice group due to enhanced infiltration of enzymatic levels of myeloperoxidase from the azurophilic granules from the neutrophils due to necrosis caused by inflammation. In contrast with the LPS + R-L treated mice group showing a subsequent decrease in MPO enzyme reaction. III. Superoxide dismutase (SOD) Assay: During LPS induction, the free radical accumulation will be more which is counteracted by the scavenging activity of the SOD enzyme. The specific activities of SOD in control and treatment groups are summarized in Fig. 5-c. The SOD tissue lysate was known to significantly increase in the LPS treated in contrast to LPS + R-L treated mice group, showing a significant decrease in SOD appropriately. Figure 5: Effect of R-L compound on different assay methods in LPS induced mice. The assay methods such as (5-a) Elastase assay; (5-b) Myeloperoxidase Assay; (5-c) Superoxide dismutase Assay were performed from the lung tissue homogenate extracted from different experimental grouping of mice. Data are represented as the mean ± SD.