Phenolic-rich fraction of green tea attenuates histamine-mediated cardiopulmonary toxicity by inhibiting Cox-2/ NF-κB signaling pathway and regulating oxidant/antioxidant balance

Background Histamine (HIS) has a substantial impact on the development of numerous allergic disorders including asthma. Antihistamines mostly target histamine receptor-1 alone, so it is not entirely effective in the treatment of allergic diseases. In the current investigation, we examine the growing evidence for novel therapeutic strategies that aim to treat histamine-mediated cardiopulmonary toxicity with the phenolic-rich fraction of green tea (PRFGT). Results Our findings demonstrated that weekly ingestion of HIS to rats induced oxidant/antioxidant imbalance in both lung and heart homogenates. The histopathological examination demonstrated extensive interstitial pneumonia with progressive alveolar and bronchial damage in HIS receiving groups. Heart sections showed severe myocardial necrosis and hemorrhage. All lesions were confirmed by the immunohistochemical staining that demonstrated strong caspase-3, cyclooxygenase-2 (Cox-2), and tumor necrosis factor-α (TNF-α) protein expressions along with upregulation of the pulmonary m-RNA expression of TNF-α, nuclear factor kappa-B (NF-κB), and interleukin-1β (IL-1β) genes and cardiac levels of many apoptotic genes. Otherwise, the pretreatment of rats with PRFGT had the ability to alleviate all the aforementioned toxicological parameters and return the microscopic picture of both lung and heart sections to normal histology. Conclusions We concluded that PRFGT’s powerful antioxidant, anti-inflammatory, and anti-apoptotic properties can reduce cardiopulmonary toxicity caused by HIS. We recommended daily intake of green tea as a beverage or adding it to foods containing elevated levels of HIS to prevent its possible toxicity.


Background
Anaphylaxis and other allergic illnesses are becoming more common everywhere, but especially in low-and middle-income nations [71].Around 250 million individuals worldwide have food allergies, while 300 million people have asthma [70].Additionally, several allergic disorders frequently occur in the same person at the same time.The main mediator of allergic diseases is histamine, and it performs all its functions via four G receptors but mostly via histamine 1 receptor (H1R) [52].Histamine is widely formed in many foods and drinks such as wine, cheese, decomposed fish, and fermented cheese via decarboxylase enzyme [35,60].The development of many allergy illnesses is significantly enhanced by histamine and its four receptors [87].Histamine is widely distributed across all cells and is found in significant amounts in the lungs, skin, and digestive tract [58].Histamine is a strong inflammatory mediator that is frequently linked to anaphylatoxins, cardiovascular alterations, and potent inflammatory reactions [19].
All green plants contain polyphenols, usually in varying amounts.In numerous experimental models, green tea extract (GTE) has a potent anti-inflammatory property [74,78,79,82], through inhibiting the gene levels of nuclear factor kappa-B (NF-κB) and interleukin-1β (IL-1β) [21,50].Another study showed that daily consumption of green tea polyphenols over 12 weeks could enhance the blood flow and oxygen supply to the skin, protecting it from UV-damaging radiation and enhancing women's overall skin quality [34].Additionally, green tea polyphenols' capacity to chelate transition metals and quench reactive oxygen species (ROS) has been found to have substantial antioxidant activity in vitro [49].Following chronic cerebral hypoperfusion, green tea polyphenols at dose of 400 mg/kg per day increase the spatial cognition because of their antioxidant properties [98].The majority of the results from research on humans showed that green tea has anti-inflammatory properties that are explained by its potent antioxidant effects that scavenge ROS and finally reduces NF-κB activity [64].Another study explained the ability of GTE to reduce doxorubicin-induced cardiotoxicity by enhancing the heart's antioxidant defenses and bringing lipid peroxidation (LPO) levels back to normal level [46].In osteoarthritis patients, GTE is used as an additional therapy to help manage pain and improve the physical function of the knee joints [28].Besides the antioxidant and antiinflammatory effects of GTE, several in vitro studies investigated its potent antiallergic effect in several cell lines via binding with immunoglobulin E (IgE) [29,97].Some fractions of green tea including epigallocatechin gallate and gallic acid have been investigated in several in vitro studies that demonstrating their ability to reduce IgE-mediated HIS release from mast cells and human basophiles [47,86].
However, the in vivo study regarding the antihistaminic effect of GTE or its fraction is missed, but it is particularly important to study the potential mechanism underlying those effects.Despite increasing the incidence of histamine toxicity worldwide, it is important to find available safe ways to reduce such toxicity.Therefore, our work aimed to investigate the possible protective properties of PRFGT against HIS-mediated cardiopulmonary toxicity.

Preparation of green tea phenolic-rich fraction
Green tea (Camellia sinensis) was acquired from the local market.The identity and purity of the green tea were verified by Mrs. Therese Labib, consultant at Ministry of Agriculture and the former director of El-Orman Botanic Garden.For column chromatography, silica gel 60 (pore size 60 Å, 70-230 mesh, 63-200 μm, purchased from Fluka, Sigma-Aldrich Chemicals, Germany) was utilized.All experiments were performed at room temperature.The extract acquired from 1 kg of green tea was applied onto a silica gel column (5 × 100 cm) and then was eluted with 85:15 dichloromethane/ethanol (v/v) followed by elution with 100% ethanol.The ethanol eluate was rich in polyphenolic metabolites and was evaporated under vacuum at low temperature to dryness [51].The residue was then kept at − 20 °C for further analysis and biological activities.

UPLC-MS/MS identification of green tea phenolic-rich fraction
The polyphenolic-rich fraction was dispersed in HPLC grade methanol at a concentration of 100 µg/ml, after which it was filtered through a membrane disk filter (0.2 m) and subjected to LCMS analysis using a UPLC/ESI-MS system with an ACQUITY UPLC-BEH C18 (1.7 µm-2.1 × 50 mm) column from Waters Corporation.10 µl of the sample was injected.We used solvent of water (A) and acetonitrile (B), each containing 0.1% formic acid.
Stepped mobile phase extraction was planned to start at 90% A/10% B for 2 min, increase to 70% A after 5 min, 30% A after 15 min, and 10% A after 22 min, all of which were to be maintained for 3 min.After 26 min, 100% B was reached and maintained for 3 min, and after 32 min, flow rate: 0.2 ml/min., the process resumed to the original composition.The study was done in negative ionization mode using an XEVO TQD triple quadrupole mass spectrometer from Waters Corporation in Milford, Massachusetts.The mass spectrometer's cone voltage is 30 eV, the capillary voltage is 3 kV, the source temperature is 150 °C, the dissolving temperature is 440 °C, and the flow rates for the cone gas and the desolvation gas are 900 L/h and 50 L/h, respectively.The ESI can detect mass spectra between m/z 100 and 1000.Metabolites were potentially identified by comparing the retention durations (Rt) and mass spectra of the peaks and spectra processed using Masslynx 4.1 software with the counterparts published in databases and literature.

Animals and experimental design
All treatments applied on rats were certified by the institutional animal care and use committee of Cairo University (IACUC) (approval number: 8032022402) and following the ARRIVE guidelines (PLoS Bio 8(6), e1000412, 2010).30-Male Wistar albino rats, weighing 170 ± 10 g, were used in this study.Five rats were housed in plastic cages and received a consistent 12 h dark/light cycle for each animal in a well-ventilated environment.They had unrestricted access to tap water during the trial as well as dry commercial standard pellets to consume.They underwent acclimatization two weeks prior to the trial's start in order to protect their health.The rats were separated into 6 groups randomly, each group consisted of 5 rats, and all rats received various treatments by oral administration for 14 days.Group (1) received distilled water.Groups (2 and 3) received PRFGT at doses 100 and 200 mg/kg BWT/day, respectively.Group (4) received HIS (98%, LOBA., India) at dose 1750 mg/kg BWT/week.Groups (5 and 6) received the identified of PRFGT + HIS as before.Both dosage levels of HIS and PRFGT were chosen according to the previous studies [13,30,44]

Sampling
After 14 days of treatment, rats were anaesthetized by intramuscular injection of Ketamine (90 mg/kg BWT) and Xylazine (10 mg/kg BWT) and then euthanized by the cervical dislocation and samples were collected from the primary target organs (lung and heart).All samples were split into two portions: One of them was promptly preserved in 10% neutral buffered formalin for histology and immunohistochemistry, and the other was stored at -80°C until it was required for redox status assessments and molecular analysis.

Measuring the tissue content of MDA, GSH, and catalase
Known weight samples from both pulmonary and cardiac tissue were homogenized with PBS (pH 7.4) and centrifuged at ×4500g.For the purpose of performing certain oxidative and antioxidant indicators, the supernatant was kept at − 80 °C.We assessed malondialdehyde (MDA), reduced glutathione (GSH), and catalase (CAT) in accordance with the guidelines provided by the manufacturer's kits (Biodiagnostic Co., Cairo, Egypt).

Histopathological examination
Following the method portrayed by Bancroft and Gamble, [10], both pulmonary and cardiac tissue specimens were washed, managed using alcohol gradients and xylene, paraffinized, sectioned into 4.5 μm thick sections, and stained with hematoxylin and eosin (H&E).
We used an Olympus BX43 light microscope to examine all stained sections and an Olympus DP27 camera connected to CellSens dimension software to capture images.A classical semiquantitative grading technique was employed following the procedures explained by Passmore et al. [69] and Hassanen et al. [32] to assess the distribution and severity of the pathological alterations within lung and heart tissues.The lung was investigated for any vascular, bronchial, alveolar, and interstitial lesions including cellular degeneration, inflammatory cells infiltration, edema, hemorrhage.Meanwhile, the heart was inspected for signs of hemorrhage, vascular congestion, interstitial edema, and muscle degeneration and necrosis.The following four-point grading scale was used to rank all the pathogenic parameters.Score (0) indicates normal histology without any microscopic changes.Score (1) indicates slight changes (tissue damage (TD) less than 10%).Score (2) indicates minor changes (TD between 11 and 25%).Score (3) indicates moderate changes (TD between 26 and 50%), while (4) means severe changes (TD greater than 50%).

Immunohistochemical staining
The apoptosis marker (caspase-3) and the inflammatory markers (Cox-2 and TNF-α) were detected in either lung or heart tissue.Briefly, the deparaffinized dehydrated tissue sections were blocked using Peroxidase (Sakura BIO) and harvested with various primary antibodies (Abcam, Ltd.), followed by reagents involved in the avidin-biotin detection system (Power Stain 1.0 Poly HRP DAB Kit; Sakura).After ten minutes of treatment with 3,3′diaminobenzidine chromogen substrate, the sections were counterstained with hematoxylin and then examined using an Olympus BX43 light microscope and photographs were taken using an Olympus DP27 camera.

RT-PCR evaluation of certain genes' m-RNA levels in cardiopulmonary tissues
Lung and heart tissues weighing about 100 mg were subjected to total RNA extraction using the ABT total RNA mini extraction kit (Applied Biotechnology, co.ltd, Egypt).RNA purity and concentration were assessed using a NanoDrop ND-1000 spectrophotometer [41].The c-DNA synthesis was performed by using ABT H-minus c-DNA synthesis kit (Applied biotechnology, co.ltd, Egypt).The m-RNA expression levels of the studied genes were detected using fluorescence-based real-time detection method according to the protocol of ABT 2X sybr mix (Applied biotechnology, co.ltd, Egypt).
Using the primer designing tool (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast/), the real-time PCR primers were created, as shown in Table 1.Each real-time PCR was done in triplicate, and the GAPDH gene was used as internal control [4,5,43].The fold change of the results was calculated from the equations of CT, ΔCT, ΔΔCT, and 2 −ΔΔCT [20,37].

Statistical analysis
The

UPLC-MS qualitative profiling of phenolic-rich fraction in green tea
A total of 52 metabolites were tentatively identified in the phenolic-rich fraction of green tea (Table 2, Fig. 1).
All the identified compounds were mainly of phenolic nature belonging to the flavonoids group the majority of which are flavan-3-ol and flavanol derivatives, in addition to phenolic acids.Flavan-3-ols, known as monomeric flavanols, include epicatechins, epigallocatechin, and their gallate derivatives.The major flavan-3-ol identified compounds in the green tea phenolic-rich fraction 21, 35, 41, and 47 at ESI − m/z 289 − , 577 − , 441 − , and 451 − generated a common product ion at m/z 289 which is a characteristic mass of epicatechin.These compounds were tentatively identified as epicatechin, procyanidin B, epicatechin gallate, and epicatechin-hexoxide, respectively.Compounds 12 and 44 at m/z 305 − and 457 showing the molecular ion m/z 305 − of epigallocatechin were identified as epigallocatechin and epigallocatechin gallate, respectively.A total of 13 flavanols were tentatively identified in the phenolic-rich fraction of green tea.The major compounds 32, 37, and 40 showing product fragment ions at m/z 285 were identified as kaempferol glycosides, namely kaempferol rutinoside, kaempferol hexosyl deoxyhexosyl hexoside and kaempferol hexosyl deoxyhexosyl hexoside, respectively.In addition to, other flavanols as myricetin and quercetin derivatives were identified from their mass fragmentation and compared to the literature as depicted in Table 2.
Ten phenolic acids (5 hydroxybenzoic acids and 5 hydroxycinnamic acids and derivatives) were identified in the phenolic-rich fraction of green tea.Gallic acid was the most abundant identified phenolic acid.The removal of the CO 2 (44 Da) and hexosyl moiety (162 Da) from their parent ions serves as a common example of the MS/MS fragmentation pattern of phenolic acids.Seven organic acids were detected as denoted in Table 2, and one polyphenol, viz.theaflavin-3,3'-digallate.

Oxidative stress evaluations
HIS receiving group displayed noticeably greater levels of MDA and lower levels of GSH and CAT than other groups.On the other side, the PRFGT-treated groups at both doses displayed significantly lower MDA content   and higher CAT and GSH content than HIS receiving group, whereas the marked improvement noticed in group receiving PRFGT at dosage level 200 mg/kg Bwt.There was no discernible difference between the HIS + high dose of PRFGT group and the control group in terms of oxidant or antioxidant levels (Fig. 2).

Histopathological examination
Compared to the control group (Fig. 3a), lung sections of HIS group displayed severe histological changes.Extremely vascular congestion accompanied by severe interstitial pneumonia was the prominent lesion in all sections.The majority of the blood vessels showed vasculitis manifested by endothelial necrosis and vascular wall thickening with inflammatory cells infiltration within and around the blood vessels (Fig. 3b).Most alveoli showed damage and others showed hemorrhage.Macrophages and eosinophils were the most noticeable inflammatory cells, along with other granulocytic cells (Fig. 3c).Moreover, there are extreme interstitial fluid and hemorrhages.The majority of the bronchi displayed epithelial desquamation, along with luminal inflammatory cells infiltration (Fig. 3d).Toluidine blue staining revealed an enormous number of dark blue/purple granulocytic mast cells in several areas especially surrounding the blood vessels (Fig. 3e).On the other side, the group cotreated with PRFGT along with HIS exhibited dose-dependent improvement in the microscopic appearance of the lung sections.Groups cotreated with 100 mg PRFGT normal histological structure of alveoli, blood vessels, bronchi, and bronchioles (Fig. 3g).However, mild thickening in the interalveolar septa by inflammatory cells infiltration was recorded in some sections along with sporadic bronchial epithelial vacuolation and necrosis (Fig. 3h).Moreover, the group receiving HIS + 200 mg PRFGT showed normal histological structure as shown in the control group (Fig. 3i-j).Table 3 provides our findings about the microscopic lesion score in lungs of diverse groups.The score for all parameters in all HIS receiving groups significantly increased in contrast to the control group.Otherwise, PRFGT-treated groups at both doses noticed a significant reduction in the pulmonary lesion scoring compared with HIS group, whereas the lowest score noticed in high-dose receiving group.In comparison with the control group, there is not a significant difference in the microscopic score of the group receiving high dose of PRFGT.
In contrast to the control groups (Fig. 4a), a significant histological change was seen in the heart sections of HIS receiving group.There were diffuse intermuscular hemorrhage and inflammatory cells infiltration commonly eosinophils, together with myocardial degeneration and necrosis (Fig. 4b, c).While group receiving HIS + PRFGT at both doses showed marked improvement in the histological appearance of cardiac muscles (Fig. 4d, e), the best improvement was observed in group receiving the high dose (Fig. 4f ).
Table 4 provides the results of the myocardial lesion scoring.The HIS obtained group showed significantly higher scores than control group and PRFGT obtained groups.Moreover, the groups receiving PRFGT at both doses showed lower scores than HIS receiving group.In comparison with those receiving the greater dose of PRFGT, the higher dose group showed a lower score.

Immunohistochemical staining
Lung samples taken from the HIS given group displayed TNF-α and Cox-2 immunopositivity stronger than other groups.On the other side, HIS + PRFGT group demonstrates dose-dependent decreasing in both immunostaining reactions.The PRFGT low-dose receiving group showed mild-to-moderate immunostaining, while those receiving the high dose did not influence any immune reactions.Heart sections obtained from HIS group demonstrated strong positive casp-3 immunoexpression.The groups cotreated with HIS and PRFGT exhibited negative to weak casp-3 immunostaining (Fig. 5).

RT-PCR evaluation of certain genes' m-RNA levels in cardiopulmonary tissues
In this study, the transcript levels of some inflammationrelated genes (TNF-α, NF-κB, and IL-1β) were measured in the lung of rats.The expression levels of all genes were upregulated in the HIS group.PRFGT ameliorated the inflammatory effect of HIS on the lung of rats but did not return to the level of the control group.The higher dose of PRFGT was more effective than the lower dose.Also, three apoptosis-related genes (c-Jun, c-Fos, and c-Myc) were measured in the heart of rats.The expression levels of the apoptosis-related genes were upregulated in the HIS group.PRFGT amended the apoptotic effect of HIS on the heart of rats but did not return to the control group level.The higher dose of PRFGT was more effective than the lower dose (Fig. 6).

Discussion
Histamine is a biogenic amine produced from the amino acid L-histidine by the enzyme L-histidine decarboxylase and is degraded by the enzyme diamine oxidase (DAO) and histamine N-methyltransferase (HNMT) [81].Despite the presence of both enzymes in the intestinal epithelium, DAO serves as the primary barrier of HIS absorption into the blood stream [76].
The ability of HNMT to breakdown HIS only exists intracellular, where it is found in the cytosol [68].Several kinds of foods and drinks contain prominent levels of HIS, including wine, cheese, fermented meat, sea food, and any decomposed fish [35].By consuming high quantities of such foods, DAO and HNMT enzyme's capacity to degrade HIS is limited, allowing it to enter the bloodstream and distribute in several organs [17].
It is distributed throughout the entire body, but lungs, skin, and digestive tract have the highest quantities [24].Moreover, some people suffer from decreased ability of the gut to break down histamine due to diminished DAO activity, which causes an accumulation of HIS in the plasma and this condition known as histamine intolerance (HIT) [17].Histamine participates in several immunological and physiological functions as well as promoting gastric secretion, inflammation, contraction of smooth muscles, vasodilatation, permeability, and many other pathological conditions [96].By increasing the incidence of food poisoning by HIS, it is important to find safe ways to prevent the risk of HIS poisoning in humans and animals.In our previous study, we explored the potential mechanism of repeated oral intake of HIS to rats.Thus, the current study was designed to assess the cardiopulmonary protective effect of PRFGT against such toxicity with comprehensive insight on the molecular mechanism.Cardiac and pulmonary tissues of all HIS receiving groups displayed a discernible raise in MDA content and a decline in CAT and GSH content, indicating the presence of oxidative stress.Reactive oxygen species (ROS) overproduction is the outcome of a redux status imbalance, resulting in significant tissue damage [90].Our histopathological outcomes revealed severe pulmonary interstitial inflammation along with myocardial degeneration because of oxidative stress.Histamine causes airway epithelial cells to produce more H 2 O 2 via signaling the H1R.The main generators of ROS are Duox1 and 2 Widening of alveolar septa 0 a 0 a 0 a 4 b 2 c 0 a that are expressed in the bronchial epithelial cells [12].These cells have the highest amounts of H1R expression, which regulates histamine's capacity to generate H 2 O 2 .Inflammatory cells like neutrophils and macrophages are released when histamine is present leading to excessive ROS generation [18].Furthermore, our results proved that mast cells and eosinophils have important roles in HIS-inducing pulmonary inflammation and cellular damage.Toluidine blue is a metachromatic stain that was largely used to identify mast cells [7], while the Congo red stain accurately diagnoses eosinophils within tissues [73].Mast cells and eosinophils play a key role in several allergic reactions including asthma and anaphylaxis [84].One of the postulated mechanisms of histamine toxicity is by stimulation of mast cells via signaling IgE and histamine receptor-1 (HR1) to release endogenous histamine and other cytotoxic mediators [36].Mast cells have the ability to regulate the activities of numerous organs and tissues via releasing the variety of multifunctional preformed molecules, including histamine, proteases, prostanoids, heparin, and numerous cytokines, chemokines, growth factors, and lipid mediators [48].The vascular endothelium can be significantly affected by these mediators, increasing the vascular permeability and adhesiveness.
All the above-mentioned aspects had the ability to bring more inflammatory cells to the localized area causing further inflammation and tissue damage [6].Moreover, eosinophils normally present in blood and other tissues such as skin, thymus, and spleen once activated by allergen (HIS) migrate to the site of inflammation.The eosinophil chemotactic factor produced by mast cells plays a significant role in bringing eosinophils to the inflamed area [61].Eosinophils also secrete cytotoxic mediators as major basic proteins, cytokines, chemokines, lysosomal enzymes, growth factors, and ROS that induce inflammation and tissue necroptotic damage [25].
The inflammatory impact of HIS is confirmed by both immunohistochemical and molecular studies which determine a strong positive expression of the inflammatory markers Cox-2, TNF-α, IL-1β, and NF-κB.The vasodilation occurs during HIS-mediated inflammation results in both exudate and inflammatory cells accumulation in the interstitial tissues [22].Additionally, HIS binds to H1R and H4R, causing generation of proinflammatory cytokines like IL-6 and TNF-α [87].TNF-α, IL-1, and IL-6 are the crucial proinflammatory cytokines that induce inflammation in many pulmonary pathologies and diseases.TNF-α is a cytokine that promotes inflammation and has a variety of biological consequences [92].TNF-α induces infiltration of the inflammatory cells, production of inflammatory mediators, oxidative and nitrosative stress, airway hyperresponsiveness, and tissue remodeling [55].The cyclooxygenase enzyme (Cox) is commonly linked to the incidence of many inflammatory disorders [91].It has been demonstrated that proinflammatory cytokines can increase Cox-2, which exacerbates the inflammatory immune response in lung damage [38].
In the present investigation, we found that the Bax/ casp-3 signaling pathway-mediated apoptosis also shared in the mechanism of cardiopulmonary toxicity that induced by HIS.Overproduction of ROS within cells damages proteins, nucleic acids, lipids, membranes, and organelles, which may trigger cell death processes including apoptosis [26].Via the mitochondrial pathway, ROS can trigger the release of cytochrome c from the mitochondria and induce apoptosis [16].In the presence of ATP, released cytochrome c interacts with apoptotic protease activating factor-1(Apaf-1) and activates caspase-9 to produce an apoptosome [9].Caspases 3 and/or 7 are then activated by an active caspase-9, which cleaves a certain set of substrates and encourages cell death [42].We found that HIS receiving groups showed strong immune expression of casp-3 along with upregulation of protooncogene genes c-fos, c-Myc, and c-Jun.These genes participate in cell cycle progression and cellular proliferation [100].A regulatory protein called c-fos contains a basic leucine-zipper region that allows it to bind to a variety of proteins [101].Both c-fos and c-Jun dimers promote the formation of the activator protein-1 Fig. 5 Photomicrograph demonstrating the pulmonary expression of both Cox-2 and TNF-α immune markers as well as caspase-3 immunostaining in the heart sections of various groups.a-c Control group exhibited negative expression of the above-mentioned immune markers.d-f Group receiving HIS showed strong immunopositivity of Cox-2 and TNF-α in the pulmonary tissue along with strong caspase-3 protein expression in the myocardium.g-i Group receiving HIS + 100 mg PRFGT displayed moderate immunopositivity of Cox-2 and TNF-α in the pulmonary tissue along with negative caspase-3 protein expression in the myocardium.j-l Group receiving HIS + 200 mg PRFGT displayed negative expression of the studied immune markers in both lung and heart section (AP-1) [40].AP-1 regulates numerous biological functions, such as cell division, cell death, survival, and differentiation [102].The c-fos gene aids in the process of myocardial apoptosis [11].It is reported that c-Jun induces and transactivates caspase-3 gene [83].One of the most crucial transcriptional factors, c-MYC, controls a wide variety of cellular processes, including apoptosis, growth, and proliferation [62].The ability of the c-Myc protein to promote apoptosis in a variety of cellular settings is one of its well-known functions [72].
Otherwise, the groups treated with PRFGT demonstrated a noticeable decrease in MDA levels and a higher antioxidant activity, indicating strong antioxidant properties of green tea phenolic-rich fraction.The presence of high concentration of flavonoids and phenolics may be primarily responsible for this action.Catechins present in green tea are famous for their anti-inflammatory, antioxidant, cardioprotective, and anticancer effects.They are the elementary unit of compacted tannins generally recognized as pro-anthocyanidins with a variety of pharmaceutical functions [4,5].EGCG is the strongest antioxidant among all catechins in green tea [88].EGCG acts as a scavenger of several ROS/RNS by capturing peroxyl radicals, and therefore, it can prevent membrane lipid peroxidation and protect cells from oxidative damage [77].Gallic acid (GA) and its derivatives are considered the primary polyphenolic compounds in green tea and also in some fruits [63].GA increases the levels of glutathione, glutathione peroxidase, glutathione reductase, and catalase as well as lowering the oxidative stressrelated damage [56].
Our study showed that PRFGT decreased the expression of Cox-2 and TNF-α immune markers and downregulated the inflammatory genes (IL-1β, TNF-α, and NF-κB) in the pulmonary tissue, indicating the antiinflammatory effect of green tea phenolic-rich fraction.One study revealed the ability GTE to reduce COX-2 activity, which attenuates lipid peroxidation and PGE2 accumulation [15].Other studies explained the anti-inflammatory potential of green tea polyphenols by regulating the COX-2 and NF-κB pathways [67,85,94] investigated that EGCG-mediated NF-κB inactivation plays a key role in its anti-inflammatory potential via regulating Cox-2 and iNOS.Green tea polyphenols exert powerful antioxidant and anti-inflammatory effects by regulating a variety of gene expressions, including Nrf2, Cox-2, iNOS, NF-κB, AP-1, and STATs [89].Previous in vitro investigation has shown that EGCG has anti-inflammatory properties and prevented neutrophil chemotaxis [8].Numerous studies have demonstrated that EGCG reduces inflammation by altering the NF-κB/ AP-1 pathway [99].For instance, the release of histamine and leukotriene B4 was significantly inhibited by EGCG [59].More studies discussed the anti-inflammatory effect of GA against several pulmonary hypersensitivity reactions which may be mediated by inactivating the transcription levels of IL-33, IL-5, and IL-13 [75,93].Recent study proved the anti-inflammatory effects of GA against toxic hepatitis via downregulating the proinflammatory cytokines, IL-1, IL-6, Cox-2, TNF-α [65].Additionally, our study revealed that PRFGT showed mild expression of caspase-3 immune marker in myocardial tissue along with down-regulation of apoptotic genes such as c-fos, c-Myc, and c-Jun which indicates the strong anti-apoptotic effect of green tea extracted fractions.This outcome agreed with the findings of Zong et al. [103], who showed that EGCG had an anti-apoptotic potential.Moreover, some studies showed that EGCG has a cardioprotective effect via significant reduction in the pro-apoptotic proteins such as Bax, caspase-9, and caspase-3 and increases the anti-apoptotic proteins such as Bcl2 [66].Another study showed that the treatment with EGCG results in mitochondrial-level cardiac protection via reversed the mitochondrial and nuclear changes caused by apoptosis [3].Furthermore, GA had a potent antiapoptotic activity through inhibition of Bax/Bcl2 ratio [14,53] and reduction of the levels of caspase-3 [57].

Conclusion
We concluded that the weekly oral intake of HIS to rats had the ability to cause severe cardiopulmonary toxicity either through oxido-inflammatory stress or apoptosis.Additionally, we found that the cotreatment of PRFGT with HIS can restore the oxidant/antioxidant balance and improve the microscopic picture of both lung and heart tissues.PRFGT had an anti-inflammatory effect via modulating Cox-2/NF-κB mediated by inactivation of proinflammatory cytokines including TNF-α and IL-1β.Furthermore, the anti-apoptotic effect of PRFGT is attributed to downregulating the casp-3, c-jun, c-fos, and c-myc genes.We found that PRFGT has a synergistic effect aid in the prevention of HIS-inducing cardiopulmonary toxicity suggesting its therapeutic potential against several HIS-mediated inflammatory diseases like atopic dermatitis, neuroinflammation, and allergies.We recommend daily intake of green tea as a beverage or adding it to foods containing elevated levels of HIS to prevent its possible toxicity.

Fig. 1
Fig. 1 Representative UPLC-MS base peak chromatogram of phenolic-rich fraction of Camellia sinensis in negative ionization mode

Fig. 2
Fig. 2 Bar graphs demonstrating the effects of HIS on various oxidant and antioxidant markers in the homogenates of cardiopulmonary tissue.a Malondialdehyde (MDA), b reduced glutathione (GSH), and c catalase activity.Means ± SEM are used to represent values (n = 5).Various superscript letters (a, b, c, etc.) indicate a significant difference between groups at P ≤ 0.05

Fig. 3
Fig. 3 Photomicrograph of lung tissue sections representing; a control group with normal histologic structure, b-f group receiving HIS, g-h group receiving HIS + 100 mg PRFGT, and i-j group receiving HIS + 200 mg PRFGT.Note: multifocal inflammatory cells infiltration (black stars), vasculitis (red stars), alveolar damage (black arrows), eosinophils infiltration (red triangles) bronchiolar epithelial necrosis (blue triangles) with luminal inflammatory cells infiltration (blue stars), interstitial hemorrhage (black triangles), mast cells aggregation (red arrows), and mild thickening of the interalveolar septa (blue arrows).All sections were stained with H&E except e stained with toluidine blue and f stained with Congo red

Fig. 4 Table 4
Fig. 4 H&E-stained heart tissue sections corresponding to; a control group with normal histological organization, b, c group receiving HIS, d, e group receiving HIS + 100 mg PRFGT, and f group receiving HIS + 200 mg PRFGT.Note: muscular hemorrhage (black stars), degeneration and necrosis of the myocardium (black arrows)

Fig. 6
Fig. 6 Bar graphs display the variations in gene transcription levels between groups in lung and heart tissues.a, b, c Indicates m-RNA levels for the TNF-α, NF-κB, and IL-1β genes in pulmonary tissue.d, e, f m-RNA levels of c-Fos, c-Jun, and c-Myc genes respectively in the cardiac tissue.Means ± SEM are used to represent values (n = 5).Various superscript letters (a, b, c, etc.) indicate a significant difference between groups at P ≤ 0.05

Table 1
Primers sequences used for qRT-PCR

Table 2
Peak assignments of metabolites in the phenolic-rich fraction of Camellia sinensis using UPLC-MS in negative ionization mode