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NARINGENIN AMELIORATES LIPOPOLYSACCHARIDE-INDUCED KIDNEY AND LUNG INJURIES IN MICE: DECISIVE ROLE OF IL-1p, IL-6, IL8, AND TNF-a
M.A. Al-Dabbagh1*, H.B. Sahib2
1 Department of Pharmacology, College of Medicine, Al Nahrain University, Baghdad, Iraq;
2 Department of Pharmacology, College of Pharmacy, Al Nahrain University, Baghdad, Iraq.
* Corresponding author: marwa.ali.ph1@gmail.com
Abstract. Cytokine storms are a major contributor to acute respiratory failure, causing numerous organ dysfunction, including kidney damage, as a result of inflammation-induced tubular injury, particularly evident in COVID-19 infections. This occurrence poses a global health risk. Experimental evidence suggests that naringenin, a natural flavonoid, has a variety of biological activities. The present study was conducted to examine the protective effects of naringenin in lipopolysaccharide (LPS)-induced lung and kidney injuries in mice and determine its relationship with the suppression of pro-inflammatory mediators. Forty-eight Swiss albino male mice were divided into four groups. Group I received 0.9% normal saline, Group II received 5 mg/kg LPS only, and Groups III and IV received 50 or 100 mg/kg naringenin, respectively, one hour before LPS administration. The effects of naringenin, vehicle, or LPS administration on mortality rate, pro-inflammatory cytokine (IL-6, IL-1, IL-8, and TNF-a) levels, and lung/renal histological changes were evaluated after 48 hours. The results showed that the naringenin-treated groups exhibited a significant (p < 0.01) reduction in pro-inflammatory cytokine levels compared to Group II. Histological examinations revealed that mice in Group II displayed significant (p < 0.01) lung and renal tissue injuries, while Groups III and IV exhibited a significant (p < 0.01) reduction in pulmonary and renal injuries, as demonstrated by improved macroscopic scores and reduced mortality. The findings of this study strongly suggest that naringenin has potent protective effects in mice against LPS-induced lung damage and associated kidney dysfunction. Consequently, naringenin has promise as a human anti-cytokine storm therapeutic agent.
Keywords: cytokine storm, inflammation, lipopolysaccharide, naringenin, lung injury, kidney injury.
List of Abbreviations
ALI - Acute lung injury ARDS - Acute respiratory distress syndrome
AKI - Acute kidney injury ANOVA - Analysis of variance b.w - Body weight CCl4 - Carbon tetrachloride COPD - Chronic obstructive pulmonary disease
COX-2 - Cyclooxygenase2 COVID - Coronavirus disease of 2019 CRS - Cytokine release syndrome CSS - Cytokine Storm Syndrome DDW - Double distilled water DMF - Dimethyl formamide DMSO - Dimethyl sulfoxide ELISA - Enzyme-linked Immunosorbent assay
ESRD - End-stage renal disease H&E - Hematoxylin and eosin IL-1^ - Interleukin-1 beta IL-6 - Interleukin-6
IL-8 - Interleukin-8 TNFa - Tumor necrosis factor a iNOS - Inducible nitric oxide synthase i.p - Intraperitoneal LSD - Least significant difference test LPS - Lipopolysaccharide LOX - Lipo-oxygenase MAPKs - Mitogen-activated protein kinase NF-kB - Nuclear factor kappa B pg/mL - Picograms per milliliter p,m - Micrometer nm - Nanometer N/S - Normal saline OD - Optical density ROS - Reactive oxygen species SEM - Standard error of the mean SIRS - Systemic inflammatory response syndrome
TLR4 - Toll-like receptor 4
Introduction
Cytokine storm syndrome (CSS), also referred to as cytokine release syndrome (CRS)
and/or hyper-cytokine syndrome, is a disorder associated with unregulated increased, circulating pro-inflammatory cytokine biomarkers that cause systemic-inflammatory clinical symptoms and severe secondary organ damage (Jar-czak & Nierhaus, 2022). It has resurfaced as a result of its association with the coronavirus of the 2020 global pandemic. Such abundant circulating pro-inflammatory cytokine/chemokine is associated with a variety of infectious and immune-mediated conditions rather than the viral load (Mangalmurti & Hunter, 2020; Mon-tazersaheb et al, 2022). Lung injury is one of the significant cytokine-release storms that could progress to serious and potentially life-threatening events. Acute lung injury (ALI) or its severe manifestation, acute respiratory distress syndrome (ARDS) is characterized by infiltration of pulmonary cells, hypoxemia, and edema (Goodman et al., 2003). On the other hand, kidney damage may cause problems with distant organs such as the lungs. However, ALI/ARDS massive alveolar epithelial damage could trigger systemic inflammation involving renal tissue damage and progress to acute kidney injury (AKI) accompanying higher morbidity and mortality rate by indirect mechanism (Salsabila et al, 2022). The major origins of AKI (such as heart failure, surgical intervention, sepsis, and hypovolemia) are linked with hypo-perfusion, shock, and ischemic damage that could cause extensive cell injury (ex. severe tubular-necrosis; Pabla et al, 2022). Although hypovolemic circulation may be the most common cause in patients, some robust proofs indicating a cytokine storm due to immune system exaggeration being the main causative factor have proven that the kidneys are also one of the key sites of disease incidence. This could lead to end-stage renal disease (ESRD) and is associated with in-hospital death (Ebrahimi-Dehkordi et al., 2021). Consequently, it gained serious clinical attention. Thus, non-classical pathways such as the development of autoantibodies that target both the kidneys and the lungs, excessive cytokine release, and stimulation of systemic inflammation may also confirm kidney-lung interaction (Fa-jgenbaum & June, 2020).
Lipopolysaccharide (LPS), an essential constituent of the Gram-negative bacteria outer membrane, and still the additional key source of systemic inflammatory response syndrome (SIRS), has been extensively utilized for establishing SIRS-related conditions mimicking the cytokine storm model in rodents as well as for exploration of drug development (Rittirsch et al., 2008; Takahashi et al, 2012; Plotnikov et al, 2018). Mechanically, LPS acts as a prototypical endotoxin by binding to the CD14/TLR4/MD2-receptor complex in various immune cell types, specifically in macrophages, B cells, dendritic, and monocytes, which stimulates the various pro-inflammatory cytokine secretion, as well as eicosanoids and nitric oxide. As a portion of the cellular stress response, superoxide is reflected as one of the crucial reactive oxygen species (ROS) brought by LPS on various immune cell types that express the toll-like receptor (TLRs) (Farhana & Khan, 2023). Lipopolysaccharide upon its TLR4-recognition, is up-regulated on bronchial epithelial cells and lung macrophages during LPS-inducing ALI. Similarly, it appears to play an important inflammatory role in AKI and is broadly expressed in leukocytes and renal epithelial cells, accordingly. It is therefore regarded as a key regulator of innate and adaptive immune- responses (Saito et al, 2005; Zhang et al., 2008). The primary effectors of LPS-in-duced systemic inflammation are pro-inflammatory cytokines, interleukin (IL)-6, tumor necrosis factor (TNF)-a, and IL1p that appear to be associated with lung/kidney injury in a cyto-kine storm. However, excessive neutrophil infiltration caused by such cytokine/chemokine is responsible for alveolar-capillary barrier injury (Ware & Matthay, 2000), resulting in the non-cardiogenic pulmonary edema as well as oxida-tive stress modulated by reactive oxygen species (ROS), which is involved in the pathogen-esis of ALI (Ward,2010). Furthermore, they could cause renal endothelial cells to secrete chemokines such as IL-8, which increase vascular permeability and cause vascular leakage, resulting in decreased renal blood flow and glo-merular filtration rate and numerous organ damage and death (Li et al., 2020; Ahmadian et
al., 2021). There is no specific vaccine or medicine for a patient with acute lung damage or kidney failure owing to the cytokine storm. Therefore, additional clinical trials are required to develop new pharmacologic derivatives with diverse biological activities.
Naringenin (4',5,7-trihydroxyflavonone) is one of the main natural flavonoids belonging to the flavone subclass, contained in citrus fruit, grapefruits, and tomatoes, the most widespread and broadly consumed vegetable crops worldwide. It is produced from phenylalanine, an aromatic amino acid. Naringenin also exists in many conjugated forms, principally as neohe-speridoside, aglycone, and glycosylated form (Shakeel et al, 2017) and it has been shown to have a variety of pharmacological and biological effects, including anti-proliferative, anti-ox-idative, anti-tumor, and anti-inflammatory properties (Shizuka et al, 2007; Li., 2015). Thus, it is considered a promising source for drug development owing to its prospective large safety margin as being derived from natural dietary sources.
The current study aimed to investigate the protective effects of naringenin at various concentrations on LPS-induced experimental renal and pulmonary damage in mice models. Correlation with the inhibition of pro-inflammatory mediators for developing a novel generation of anti-cytokine storm remedies was also investigated.
Materials and Methods
Source of animals
Forty-eight Swiss adult albino male mice (25 ± 5 grams; age: 8-10 weeks) were used in the current investigation. The animals were obtained from the Animal House Facility, National Center for Drug Quality Control and Research in Baghdad, Iraq. The animals were kept in the animal house of the Biotechnology Research Center, Al Nahrain University; Baghdad, Iraq in standard plastic cages at 22 °C ± 2 °C, relative humidity, and the air of the room was changed continuously by using a ventilating vacuum under a 12 hours light/dark cycle. All the animals were allowed free access to food and tap water (ad libitum). The mice were
allowed two weeks to acclimate to the animal house environment before the commencement of the experiment.
Ethical approval
All animal care and experiments were carried out in strict accordance with the recommendations of the Animal Ethical Committee at Al-Nahrain University, College of Pharmacy, under issue number PH-Nah 4. The experimental protocol was approved by the Institutional Review Board (IRB) of the College of Medicine, Al-Nahrain University, Baghdad, Iraq.
Preparation of naringenin working solution
Naringenin (Hangzhou Hyperchem Limited, China, CAS No.480-41-1, HPLC > 98.00% purity) working solution (50 and 100 mg/kg) was prepared by dissolving freshly prepared dimethyl formamide (DMF; BDH Chemical Ltd, Poole, England) solvent, then diluted with sterile double distilled water (DDW) and left for 1 hour. Before LPS treatment, a single dose (i.p [intraperitoneally]) was used to determine a dose that demonstrated effective survival in mice after 48 hours.
Induction of lung and kidney injury in a mouse model
A working solution of LPS was first prepared. The LPS stock solution (Hangzhou Hy-perchem. Limited, China; B.No.: 0000114326) was prepared by dissolving lyophilized powder of LPS in 0.9% normal saline (Pioneer, Iraq) and was reconstituted to the concentration of 1 mg/mL in a glass tube. It was then vortexed for thirty minutes before each usage. For a 5 mg/kg, i.p) dose, approximately 125-140 pi of the solution was injected intraperitoneally into each mouse based on body weight. To accurately calculate the dosage of LPS, healthy animals were weighed before each injection (Fig. 1).
Monitoring of experimental mice
Mice were observed every 2 hours post LPS injection up to 48 hours for the occurrence and severity of endotoxemia. The principal investigator of the current study and a veterinarian
Fig. 1. Overview of the experimental protocol for cytokine storm induction in animal models
(Adapted from BioRender.com)
from the Animal Care and Veterinary Services (Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq) jointly evaluated the animals utilizing variables that have been designated in the previous literature and as follows:
1. Checking the appearance of the fur, which is usually smooth, ruffled, spiky, or puffy fur, and shows, discomfort and/or pain.
2. Checking the activity of the mouse: Mice were generally easily moved across the cage, eating, drinking, and ascending, but restricted activity could be a symptom of pain.
3. Checking the consciousness level: Mice were very snoopy, and they normally moved around the cage inspecting the surroundings. Lack of or reduced movement indicated pain and discomfort.
4. Checking the eye condition: Fully opened eyes were anticipated in apparently healthy mice, but partly or fully closed eyes, probably with secretion, could be an indication of pain.
5. Checking the respiratory quality: Difficulty in breathing with/without gasping is a sign of pain.
6. Checking the respiratory rate: Healthy mice frequently have a regular and rapid respiratory rate, while reduced respiration might be a sign of distress.
Grouping of experimental mice
The forty-eight mice were assigned at random (to avoid experimental bias) into four subgroups (n =12) after being left for two weeks for acclimatization. Group I (control group) contained twelve mice that received 0.9% normal saline vehicle i.p. In Group II, twelve mice received 0.9% normal saline vehicle and LPS lipopolysaccharide (5 mg\kg, i.p, b.w) only (Ramirez et al, 2019). Group III was made up of twelve mice that received 50 mg/kg, i.p, b.w of naringenin, then within one hour, 5 mg\kg, i.p, b.w of LPS were administered through injection. In Group IV, twelve mice received 100 mg/kg, i.p, b.w of naringenin, then within one hour, 5 mg\kg, i.p, b.w of LPS were injected.
Sample collection
Forty-eight hours after the LPS administration, all mice were anesthetized with diethyl ether (BDH Chemical Ltd, Poole, England) by light anesthesia (Kaela et al, 2021), before being sacrificed by cervical dislocation. Then blood, lung, and renal tissue samples were collected.
Collection and preparation of blood samples
Blood samples were obtained by using the direct cardiac puncture technique (Parasuraman
et al., 2010) from each mouse before death, in which about 0.5-1.5 ml of blood was collected. To collect serum, extracted blood was placed in a gel tube for about 20 minutes until clotting occurred, then centrifuged for about 10 minutes at 10000 rpm. The collected serum was centri-fuged at 3000 x g/sec for 1 minute to remove red blood cells (RBCs). The obtained samples with significant hemolysis were discarded. The supernatant was stored at -20 °C for further analysis.
Measurement of inflammatory markers
Following the national security protocols of biological laboratories, the levels of inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-1p (IL-1P), interleukin-6 (IL-6), and interleukin-8 (IL-8) levels in the collected serum of mice were determined after the experiment. The measurement was conducted using the sandwich-enzyme-linked immuno-sorbent assay (ELISA) approach with a commercial kit (BioSource, USA) following the manufacturer's instructions. At 450 nm, the absorb-ance was measured using a microplate reader (Bio-Tek Instruments, Inc., USA). The sample concentrations were calculated with a standard curve and the results were expressed as pg/ml. The experiments were replicated three times.
Histopathological analysis
Forty-eight hours after LPS administration, and immediately following the scarification procedure, histological examinations on mouse-extracted lung and kidney slices were conducted. Five animals from each group were randomly chosen for histopathologic evaluation of their right organ at various times. The 10% buffered formalin (Fluka Co., Switzerland) was used for fixing the lung and kidney tissues, and then dehydrated with alcohol. After overnight fixation, paraffin was embedded, cleared, and infiltrated. As a result, the tissue samples were placed in molds and waxed until they were hardened. Temporarily, tissue sections were partitioned into 4-5 pm thick and the sectioning was performed with the aid of a microtome (Sa-kura, Japan). Hematoxylin and eosin (H&E; Thermo Shandon, USA) stain was used to ex-
amine morphology and inflammatory infiltration under a light microscope (Carl Zeiss Microscope), as previously described. A Zeiss imager M2 microscope (Carl Zeiss Microimaging) fitted with an Axio-CamHRc CCD camera was applied to observe histopathological changes. The slides for lung and renal sections were coded, randomized, and evaluated by professional pathologists who were not part of the study group. The grading was done as follows:
A. The grade of lung tissue injury that was assessed based on the following histological changes: edema, inflammatory cell infiltration, interstitial inflammation, alveolar wall damage, and congestion.
B. The grade of kidney tissue alterations and severity of tubulointerstitial injuries was recorded as renal sections were arbitrarily alienated into 3 sections: the cortex, inner, and outer medulla. In each zone, the level of tubular cast formation, tubular dilatation, and degeneration (vascular changes, injury of brush border, tubular epithelial cells detachment, and condensations of tubular nuclei) was evaluated semi-quantitatively.
Assessment of macroscopic tissue scores
Histologic sections for each animal were assigned a semiquantitative average score value as the histological index of lung injury was recorded (Matute-Bello et al, 2001). For renal tissue, the injury was scored according to Nomura et al. (1995) criteria with some modifications (Table 1). The resulting scores were summed to indicate the rank of organ injury. Finally, statistical analysis and graphs were plotted according to the study scoring outcomes.
Statistical analysis
The data were expressed as the quantitative mean ± standard deviation (SD). Statistical analysis was performed using the statistical analysis system (SAS, 2018) to detect the effect of the different factors in the study parameters. The analysis of variance (ANOVA) and least significant difference (LSD) tests were utilized to compare between means. A Chi-square test was employed to compare the levels of significance (0.05 and 0.01 probability) between the percentages in the current study.
Table 1
Lung and tubular damage scoring system
Tissue findings Score
0% = no damage Grade 0
0-30% = mild damage Grade 1
30% - 70% = moderate damage Grade 2
70-100% = widespread/severe damage Grade 3
Table 2
Clinical observations
Clinical criteria Description
1. General appearance of fur Piloerection of hair; might or not exist, as the mouse seems "puffy".
2. Consciousness level Activity was impaired. The mouse only moves when triggered. With possible tremors.
3. General activity The mouse is inactive with occasional inspective movements.
4. Responses to stimulus Sluggish or no responding to auditory stimulus; or tactile stimuli. (no locomotion)
5. Quality of respiration Labored with recurrent gasps
6. Respiratory rate Considerably reduced respiration (rate not assessable by eye)
7. Eye condition Eyes were half closed or more, perhaps with secretions
Results
Survival analysis
All animals receiving (0.9% normal saline [N/S]) survived until they were killed. The mortality rate of the 5 mg\kg LPS only-injected mice (group II) was 66.67% (8/12 mice) in the untreated mice after 48 hours of LPS administration. The mortality rate was significantly (p < 0.01) reduced to 16.67% (2/12) with the 100 mg/kg naringenin treatment compared with untreated animals. The decrease in mortality rate was also achieved in mice administered with 50 mg/kg naringenin to 25% (3/12). However, there was no significant difference between naringenin-pretreated groups (III and IV) as shown in Fig. 2. Although the former had a better chance of survival.
In the naringenin (50 and 100 mg/kg) pre-treated groups III and IV, respectively, before LPS injection, the death rate was suppressed significantly to 25% and 16.7%, respectively. All mice in group I (0.9% N/S) survived. The data representing the proportion of dead animals (numbers recorded above bars) (p < 0.01) comp-
ared to group I. A high significant difference (p < 0.01) was observed compared to group II.
Description of mice at the end of the experiments
According to the results presented in Table 2, all untreated mice in the LPS-induced group demonstrated reduced activity, were lethargic, and clustered together.
Effects of naringenin on inflammatory cyto-kine levels
Collected serum of Swiss albino male mice that received 5 mg/kg LPS only (group II) after 48 hours revealed significantly elevated IL-6, IL-1p, IL-8, and TNF-a serum levels, which were approximately 6-6.5-fold compared to those of group I, that received 0.9% N/S (Figs. 3-6). To find out the impact of different naringenin doses (50 and 100 mg/kg) on the inflammatory response that occurred 48 hours after LPS administration in mice model, the in flammatory cytokine serum levels including IL-6, IL-1p, IL-8, and TNF-a were measured
Fig. 2. Effect of lipopolysaccharide only (group II) on mortality rate, 48 hours after single dose (5 mg\kg) injection (n=12)
with ELISA technique. It is worth noting that pre- treated group with naringenin at both doses revealed that serum levels of TNF-a, IL-8, IL-6, and IL-1P were reduced significantly (p < 0.01) in groups III and IV, respectively, compared to group II. Naringenin-pretreated mice at a dose of 100 mg/kg
(group IV) revealed no significant changes in the reduction of IL-8, IL-6, and IL-1P) serum levels, while highly significant (p < 0.01) reduction was observed in TNF-a serum level compared to group III (pretreated with naringenin at a dose of 50 mg/kg) and in a dose-dependent manner (Figs. 3-6).
Fig. 3. Mean serum level of TNF-a (pg/mL) in different study groups (n = 12). Group I: healthy control (received 0.9% normal saline); Group II: injected with lipopolysaccharides (LPS) only; Group III: received 50 mg/kg naringenin one hour before being injected with LPS; Group IV: was given 100 mg/kg naringenin one hour before being injected with LPS; The data were expressed as mean ± standard deviation (SD); P < 0.01: highly significant difference compared with group I (healthy control) and also highly significant difference compared with group II (the LPS only treatment)
Fig. 4. Mean serum level of IL-6 (pg/mL) in different study groups (n=12). Group I: healthy control (received 0.9% normal saline); Group II: injected with lipopolysaccharides (LPS) only; Group III: received 50 mg/kg naringenin one hour before being injected with LPS; Group IV: was given 100 mg/kg naringenin one hour before being injected with LPS; The data were expressed as mean ± standard deviation (SD); P < 0.01: highly significant difference compared with group I (healthy control) and also highly significant difference compared with group II (the LPS only treatment)
Fig. 5. Mean serum level of IL-8 (pg/mL) in different study groups (n = 12). Group I: healthy control (received 0.9% normal saline); Group II: injected with lipopolysaccharides (LPS) only; Group III: received 50 mg/kg naringenin one hour before being injected with LPS; Group IV: was given 100 mg/kg naringenin one hour before being injected with LPS; The data were expressed as mean ± standard deviation (SD); P < 0.01: highly significant difference compared with group I (healthy control) and also highly significant difference compared with group II (the LPS only treatment)
Group I Group II Group III Group IV
Fig. 6. Mean serum level of IL-1ß (pg/mL) in different study groups (n = 12). Group I: healthy control (received 0.9% normal saline); Group II: injected with lipopolysaccharides (LPS) only; Group III: received 50 mg/kg naringenin one hour before being injected with LPS; Group IV: was given 100 mg/kg naringenin one hour before being injected with LPS; The data were expressed as mean ± standard deviation (SD); P < 0.01: highly significant difference compared with group I (healthy control) and also highly significant difference compared with group II (the LPS only treatment)
Histopathological observations of pulmonary tissues
The histological investigation on pulmonary tissue was directed to provide more evidence about the effect of naringenin on LPS-induced en-dotoxemia and mediated experimental lung injury. As presented in Fig. 7A, specimens from group I revealed a typical lung tissue histological appearance, and there were no pathological findings detected (grade 0). Intraperitoneal LPS injection at a dose of 5 mg/kg caused diffused damage, alveolar destruction, and marked vascular congestion, concomitant with massive infiltration of inflammatory cells and interstitial edema. Hence, the LPS receiving group II displayed a significantly (p < 0.01) greater histopathological score of 3 than the control group I (Fig. 7B). However, pre-treatment with naringenin (50 or 100 mg/kg) groups III and IV, respectively, ameliorated these histopathological observations. Moreover, naringenin at a dose of 100 mg/kg resulted in a significantly improved histological pattern and significantly lower microscopic scores (grade 1) in group IV compared to the half dose (50 mg/kg) as displayed in Figs. 7C and D).
Histopathological observations of renal tissues
As shown in Fig. 8A, the histopathological outcome of renal tissues stained with H&E revealed normal histology in the 0.9% N/S treated group I. Meanwhile, in the LPS-treated group II, the proximal convoluted tubules were predominantly influenced by dilation, congested vessels, extensive tubular epithelial desquamation, notable inflammatory cell infiltration, and intraluminal cast formation. In addition to tubular epithelial swelling and vacuolar degeneration (Fig. 8B), the renal score of tubular injury was correspondingly elevated, accordingly. The score of tissue injury reached 3, indicating about 70-100% of the renal tissue being affected. On the contrary, both naringenin doses (100 and 50 mg/kg), respectively, reduced the LPS-inducing renal tissue injury. It is worth noting that pre-treatment with naringenin at a dose of 100 mg/kg exhibited a more reno protective effect with a histological profile almost similar to group I compared to the lower dose (50 mg/kg) as displayed in Figs. 8C and D).
Fig. 7. Histopathological observations of lung tissues stained with hematoxylin and eosin. A. Group I (Control; 10x), which was treated with 0.9% N/S, showed normal histology of the lung consisting of alveoli bordered by normal alveolar septate (blue arrow) with no infiltration of inflammatory cells (no tissue damage; score 0); B. Group II (LPS-injected only; 20x) showing heavy infiltration of inflammatory cells (green arrow), obvious vascular congestion (black arrow), diffused alveolar damage and intra-alveolar suppurative inflammation rich in neutrophils (red arrow) (severe tissue damage; score 3); C. Group III (LPS-injected and pre-treated with 50 mg/kg naringenin; 10x) showing some areas of reduced inflammatory cell infiltration and reduced inflammation (black arrow), reduced congestion (blue arrow; moderate tissue damage; score 2); D. Group IV (LPS-injected and pretreated with 100 mg/kg naringenin; 10x) showing reduced inflammatory cell infiltration in some areas, mild inflammation (yellow arrow), mild edema (red arrow), mild congestion (black arrow; mild tissue damage, score 1)
Fig. 8. Histopathological examinations of the kidney tissues stained with hematoxylin and eosin. A. Group I (Control; 10x), which was treated with 0.9% N/S, showed a normal histological appearance comprising of proximal and distal convoluted tubules (black arrow), glomeruli (blue arrow), with no inflammatory cell infiltration (no tissue damage; score 0); B. Group II (LPS-injected; 40x) showing marked distortion of kidney architecture, tubular epithelial swelling of cells and vacuolar degeneration (red arrow), dilated and congested vessels (green arrow), inflammatory cell infiltration and tubular cast formation (sever tissue damage, score 3); C. Group III (LPS-injected and pretreated with 50 mg/kg naringenin; 20x) showing moderate degenerative changes of renal tubules (blue arrow), moderate congested and dilated glomerular capillaries, reduced inflammatory cell infiltration in some areas (moderate tissue damage, score 2); D. Group IV (LPS-injected and 100 mg/kg naringenin; 20x) showing reduced infiltration of inflammatory cells (yellow arrow), and mild congestion (blue arrow; mild tissue damage; score 1)
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Fig. 9. The pathological scores of representative lung and kidney samples in each group. Data were expressed as mean ± SD, n = 5; The graph revealed that group II (LPS-injected) produced a highly significant (p < 0.01) degree score of injury compared with group I (healthy control). Meanwhile, the naringenin pre-treated groups III and IV at doses 50 and 100 mg/kg, respectively, one hour before receiving LPS showed highly significant (p < 0.01) enhancement of histopathological variations compared with group II (LPS-injected)
Discussion
Although lung and kidney injuries are distinct entities, they frequently coexist and frequently interact in severely ill patients. With a mortality rate of over 40%, most patients in the intensive care unit will die from numerous rather than discrete organ failures (Vincent, 2011). Cytokine storm syndrome is the detrimental prominent core of multi-organ failure, predominantly the lungs and kidneys, as it is characterized by systemic hyper-inflammatory conditions, as well as endothelial damage, oxi-dative stress, mitochondrial dysfunction, and apoptosis. All these are complications in the injury mechanism(s) of such organs (Gomez et al, 2014; Moreira et al, 2014). The current study focused on evaluating the prophylactic effects of naringenin in the lung, and associated dysfunctioning kidney injury after 48 hours of LPS administration on male mice by targeting the exaggerated inflammatory response while assessing the anti-cytokine storm activity via modulating the inflammatory biomarker outcomes, and their decisive role in addition to evidence of histopathological findings.
In terms of animal welfare, current study findings successfully reflected changes during endotoxemia, as well as assess its severity on a
mouse model based on symptoms identified in humans. In addition, it is considered reliable in an i.p. injection with the LPS administration) as reported by Huet et al. (2013). Researchers have utilized an endotoxin-treated mouse approach as an efficient strategy for inducing lung and kidney injuries in animal models by triggering inflammatory response pathways (Liu et al, 2019; Sahib et al, 2022). In human beings, a clinical pattern of outcomes termed the systemic inflammatory response syndrome (SIRS) describes a resident in danger of lung and kidney injuries (Pietrantoni et al., 2003; Zheng et al, 2019). The term SIRS refers to the systemic release of a variety of proinflammatory cyto-kines/chemokines that have been implicated in the pathophysiology of ALI/AKI in humans (Takala et al., 2002). Although intraperitoneal LPS injection significantly increased pro-inflammatory cytokines (IL-1ß, IL-8, IL-6, and TNF-a) in the current investigation to approximately 6-6.5-fold compared to apparently healthy control. Several lines of evidence suggested that cecal ligation and puncture could up-regulate such cytokines upon inducing an experimental ALI/ AKI animal model in a similar pattern (Bhargava et al., 2013; Zhou et al., 2019; Raheem et al, 2022; Qiongying et al,
2023). The results of the remarkable elevation of TNF-a, IL-1p, IL-6, and IL-8 serum levels in group II may be attributed to the capability of LPS linked to its unique LPS-binding protein (LBP), forming a complex of LPS/LBP. At that point, it triggers the stimulation of the CD14/TLR4 receptor on the macrophages and monocytes (a TLR4 dimer binding to the MyD88 proteins), thereby stimulating the downstream of the nuclear factor kappa light chain kappa factor (NF--kB) and mitogen-acti-vated protein kinase (MAP kinases) signaling pathways (Kolb et al, 2001; Vellaisamy et al., 2015). Subsequently, there is an over-expression of various pro-inflammatory cytokines such as TNF-a, IL-8, IL-6, and IL-1 p (Mansoor & Raghif, 2022; Farhana & Khan, 2023).
Pro-inflammatory cytokines/chemokines, such as TNF-a, IL-8, IL-1p, and IL-6, could aid the host defense and/or paradoxically induce inflammatory tissue injury. Among various pro-inflammatory mediators accountable for the amplification and transduction of the inflammatory responses is TNF-a. It has been strongly linked to the development of lung injury (Lu et al, 2012), as it directly ameliorates lung damage characterized by increased edema, permeability, and neutrophil entrapment in the micro-vasculatures. Meanwhile, IL-1p initiates the deterioration of the lung's epithelial and endothelial cellular layers (Negri et al, 2002). Thus, TNF-a and IL-1p together could induce bronchial epithelial cells to secrete IL-8, which is further increased by LPS as well. Hence, IL-8 plays an essential role in modifiable leukocyte trafficking in pulmonary illnesses, such as asthma, COPD, and ARDS (Okusawa et al, 1988; Ortiz et al., 2001; Pease & Sabroe, 2002). IL-8 is regarded as a critical chemoattractant for monocytes/macrophages, neutrophils, and T-lymphocytes during renal inflammation. Moreover, the chemokine initiates local infiltration of monocytes/macrophages and subsequently its activation (Yu et al., 2015), which further increases lung and kidney injuries induced by LPS administration, as presented in the current study's observations.
On the other hand, IL-6 plays a crucial role in the pathogenesis and progression of ALI. The
elevated level of IL-6 in the lung tissues is due to its vital role in the phosphorylation of activator and signal transducer of transcription 3 (STAT3), resulting in the enhancement of neutrophils recruitment and lowering endotoxin stress (Zhao et al., 2016). Moreover, other signal transduction pathways that comprise JAK/STAT, NF-kB, as well as mitogen-acti-vated protein kinases (MAPK), also contributed to mediating lung/kidney hyperactive inflammatory disorder (Fang et al, 2007). Thus, IL-6 is reported to be intensely involved in an inflammatory cascade of LPS-induced lung and kidney injuries as evidenced in the results of the current study. Conversely, the significant reduction in TNF-a, IL-ip, IL-8, and IL-6 serum levels in groups III and IV (pretreated groups with the naringenin at different doses) may be due to multiple pharmacological and biological effects, such as anti-oxidative, anti-inflammatory, anti-proliferative, and anti-tumor, as well as immune-modulating activity that the phyto-chemical has (Alberca et al., 2020). Furthermore, naringenin treatment inhibits inflammation-producing mediators such as inducible nitric oxide synthase (iNOS), lipo-oxygenase (LOX), and cyclooxygenase 2 (COX-2), which produce thromboxane A2 and prostaglandin E2 that could worsen cytokine storm (Park et al, 2012). Scientific evidence shows that naringenin treatment prevents the STAT-phos-phorylation, thus blocking the JAK/STAT inflammatory signaling pathway that is responsible for most cytokines signaling and growth factors affecting many essential cellular functions and promoting growth, differentiation, cell proliferation, migration, apoptosis, immune response, and inflammation (Lingtao et al, 2017).
In the survival experiment, the animals in group II that were exposed to the inflammatory stimulation had a significantly higher mortality rate than the animals in group I that were given saline. This increased mortality may be connected to a strong SIRS induced by LPS, resulting in various organ dysfunctions. However, pretreatment with naringenin at doses of 50 and 100 mg/kg for one hour before LPS administration resulted in the greatest reduction in mortal-
ity rate. Built on this outcome, the current study team predicted that naringenin at a dose of 100 mg/kg might act as an immune-modulatory agent, inhibiting the LPS biological toxicity, suggestive of providing an innovative therapeutic approach for pulmonary/renoprotective activity. Thus, naringenin specifically promotes cytokine trafficking and degradation and improves the survival capability of naringenin-pretreated mice, hence, providing a modality for drug discovery in this field.
According to the pathophysiological aspect, cytokine storms can result in oxidative stress injury, deregulated inflammatory response, and severe histological changes. Hence, histopathological assessment reinforced the outcomes obtained in the current study. According to earlier reports, the histological sign of the LPS-induc-ing group of animals had a high score value of 3 (serious tissue damage) on the previously indicated lung/kidney scoring system. The typical symptom in response to systemic as well as local inflammation is edema. Pretreatment with naringenin improved the pulmonary histo-pathological changes and attenuated the severity of lung edema significantly. Moreover, neu-trophil infiltration, cell structure damage, and thickening of the alveolar wall in the pre-treat-ment group with naringenin significantly improved the renal histopathological alteration to mild changes only (rank 1) compared with those in the LPS-induced lung injury groups.
LPS stimulation of the renal tubules considerably increased the formation of reactive oxygen species (ROS) and thus the stimulation of MAPKs. However, ROSs are produced by infiltrating cells in injured kidneys or endogenous sources and producing AKI and/or increasing renal damage (Kang et al, 2005; Aal-Aaboda et al, 2021). Hence, ROS endogenous scavengers could ameliorate vascular or renal tubular injury (Schrier & Wang, 2004; Shi et al., 2021). However, oxidative stress promotes the activation and recruitment of residential neutrophils and macrophages, prominent to hyper-inflammatory responses and thus cellular injury. The results of the current study and several evidence specified that extracts of citrus fruit and naringenin have the prospective for suppressing
oxidative stress, metal chelating, and anti-free radical scavenging activity and improving the kidney's antioxidant defense system (Jasemi et al, 2022; Zhang et al, 2022; Alaqeel & Hariri, 2023). However, in comparison to the current study findings, only a few reports have revealed citrus naringenin's renoprotective activity. It effectively protects against CCU-inducing renal injury at doses of 20 and 40 mg/kg in rats by reducing creatinine, urea, and uric acid serum levels in a dose-dependent manner (Ammar et al., 2022). Hence, alleviating inflammation and oxidative stress pathways could preserve normal lung and kidney functions and prevent histopathological changes (Abu-Raghif et al, 2015; Al-Saedi et al 2015; Ahmad et al., 2022).
Conclusion and recommendations
The findings of the current research show that the kidney plays an important role in the elimination and synthesis of mediators of lung injury and that prolonged exposure to mediators contributes to pulmonary injury. Furthermore, the study highlights the importance of the kidney in the regulation of systemic inflammatory cytokines/chemokines and pulmonary homeo-stasis. The naringenin's pulmonary/renoprotec-tive effects were observed at a dose of 100mg/kg, which was more significant compared to 50 mg/kg. This observation could be due possibly to its anti-inflammatory effect and significantly improving the survival rate. Furthermore, these phytochemicals have a pulmo-nary/nephroprotective effect by decreasing his-tological alterations caused by LPS administration and improving microscopic scores. Additional research with a more comprehensive study is required to fully understand the molecular mechanism(s) underlying naringenin's protective action against LPS-inducing in an experimental pulmonary/renal injury model or, ideally, in clinical investigations.
Acknowledgments
The authors thank Prof. Dr. Salah Mahdi Mohsen, Biotechnology Research Center, Al-Nahrain University for sharing his experience in handling animals, and for technical assistance with great support during this work.
Authors' contributions: both authors developed, conceptualized, and carried out the experiments, as well as writing the first draft of the manuscript, data analysis, and manuscript proofreading. They both agreed to be completely accountable for ensuring the integrity
and precision of this study, as well as reading and approving the final manuscript.
Funding: the authors have not specified any grant for this research from any commercial, public, or not-for-profit funding agency.
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