**3. Discussion**

LPS is a significant component of Gram-negative bacteria's outer membrane and can induce inflammatory reactions by penetrating into the bloodstream that can trigger pain and eventually to death [14]. In the current investigation LPS caused impairment of kidney and liver function markers used for the determination of respective organ injuries. This increase in biochemical markers by LPS has been previously reported [15]. LPS has been suggested to generate NO and other ROS which causes peroxidation and cell alterations which further leads to the disruption of cell membrane and subsequent release of cytosolic contents [16]. Moreover, albumin levels were decreased in the in the present investigation which are corroborated with the reports of Amin et al., 2020 [12]. Bilirubin levels in LPS-induced animals were increase 4 fold compared to the normal control animals. ZIN treatment markedly mitigated the alterations induced by LPS by diminishing the levels of CK, Scr, alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST), blood urea nitrogen (BUN), and bilirubin (BIL) and increasing levels of albumin which is supported by the previous results of Amin et al., 2020 [15].

LPS treatment causes increase in the circulating nitrite levels in LPS intoxicated rats as compare to normal control animals. It is well known that LPS causes' significant increase in the nitrite levels because of over expression of inducible nitric oxide (iNOS) [17]. The endothelial cells are responsible for the increasing circulating levels of NO at the site of infection in response to pathogen [18]. NO is producing a potent oxidant peroxynitrite (ONOO−) by reacting with superoxide anion (O2−) which increases the lipid peroxidation and causes oxidative damages in di fferent tissues [19]. The ZIN treatment was found to have protective e ffect against LPS-induced inflammation as evident by the significant decrease in circulating NO levels. These results were in agreemen<sup>t</sup> with the results of Alkharfy and colleagues in 2015, reported that thymoquinone significantly decrease the plasma levels of nitric oxide and the survival in animal model of sepsis [20]. Oxidative stress is characterized as an imbalance between free radical development and antioxidant defense. Superoxide (02−), hydroxyl (OH−) and nitric oxide (NO−) are among the most common reactive oxygen species. Antioxidants immediately reverse oxidative stress by enzymes, such as GSH, and MPO as well as by plant derived flavonoids [21], GSH (tripeptide thiol), found in cells is the most important anti-oxidant molecule and serves as a protecting agen<sup>t</sup> against pathogen induced ROS and RNS [22] and regulates the cell proliferation, and apoptosis [23]. GSH depletion is an important pathological event in many tissues [24]. MPO is an enzyme located predominantly in the primary neutrophil granules and its key function is to destroy the pathogens, although in some circumstances it yields high amount of oxidants which further leads to tissue damage [25]. ROS destroys the cell membrane, DNA damage to cells, causing oxidation of lipid bilayer releasing MDA as the end product [26,27]. MDA, a lipid peroxidation marker used to evaluate the lipid peroxidation due to oxidative stress [28]. ZIN has an exceptional ability of scavenging reactive oxygen species (ROS), free radicals and other damaging oxidants by inhibiting the enzyme xanthine oxidase [13]. Zingerone has also know to exert beneficial e ffect and protects DNA damage against stannous chloride induced oxidative damage [13]. Zingerone administration has been shown to suppress the mitochondrial injury and lipid peroxidation and mitigates proapoptotic proteins like BAX, and caspases [29]. These findings sugges<sup>t</sup> that zingerone is a potent antioxidant, which was further supported by the fact that ZIN had shown high antioxidant potential as compared to the ascorbic acid [13]. ZIN has shown antioxidant activity against superoxide and NO generated peroxynitrite causing damage to the cells [30]. The plasma levels of these oxidative markers were markedly increased in LPS-induced rats and were significantly reduced with ZIN treatment, suggesting the anti-oxidant potential of ZIN [2,15]. 8-OHdG is one of the principal forms of free radical-induced DNA damage by oxidation, thus been extensively used as an oxidative stress DNA biomarker [31,32]. The increased levels of 8-OHdG were suggested that DNA oxidation was aggravated by LPS administration. ZIN treatment has been found to prevent the LPS-induced oxidative DNA damages as evident by the decreased levels of 8-OHdG.

Elevated cytokines are recognized as inflammatory biomarkers in endotoxin-related pathogenesis [33,34]. TNFα, IL-β, ILα, IL-2, IL-6, and IL-10 are known as the key mediators of inflammation and TNFα among them is considered to be a principal cytokine regulator [35,36]. Many research studies have identified the function of TNFα and IL-1 in systemic inflammation, including both animal models of septic shock and in human sepsis trials [37,38]. Once released both TNFα and IL-1 target on di fferent cells e.g., macrophages, neutrophils and endothelial cells. TNFα contributed to improved macrophage development which stimulates macrophage activation, di fferentiation and survival of these cells and thus enhances proinflammatory responses in infection [39]. TNFα and IL-1 enhance inflammatory cascade by activating macrophages to release certain proinflammatory cytokines such as IL-6 and IL-8, ROS/RNS, and lipid mediators which are essential to sepsis-induced organ failure [40]. Increased IL-2 plasma levels with gram-negative bacteria can serve as a septic shock prognostic catalogue. IL-2 receptors are often released from T and B lymphocytes in biological fluids and tend to contribute to sepsis pathogensis. Inflammatory response of IL-6 is likely to be an e ffective mediator [41]. Endogenous IL-10 is an important anti-inflammatory cytokine, prevents animals from death in sepsis and has been recognized as a key approach for dealing several inflammatory disorders [42]. IL-10 has been reported to be beneficial in various inflammatory diseases other than sepsis or systemic inflammation such as inflammatory bowel disorder, arthritis, sclerosis [41]. In the present study, ZIN treatment to LPS treated rats showed the marked reduction in plasma cytokine levels significantly as compared to LPS treated rats. It has also been observed that the treatment with ZIN in normal control rats does not alter the physiological state. PCT is deemed one of the best biomarkers for sepsis and endotoxemia [43]. LPS toxicity is followed by its binding to lipopolysaccharide binding protein (LBP), which facilitates binding to the CD14 co-receptor, activating cell responses through TLR4 signaling. Reducing the circulating endotoxin in animals handled with ZIN indicates this flavonoid has the potential to enhance LPS clearance. Endotoxin removal occurs in the liver Kup ffer and parenchyma cells where it is catabolized. The involvement of ZIN against LPS-induced inflammation and oxidative damage in governing this protective activity further supports this possibility.

LPS-induced rats exhibiting enhanced levels of biochemical parameters were accompanied by enhanced pathological alterations of brain, lung, liver, and kidney. Staining of the brain cortex in the LPS group showed degenerated neurons with hyperchromatic nuclei and increased vacuoles which were prevented by ZIN treatment. The hepatic tissues from the normal control animals exhibited normal cellular and lobular architecture. Liver tissue from the LPS group exhibited prominent pathological alterations comprising widespread portal inflammation, hepatic cell necrosis, and infiltration of inflammatory cells. However, ZIN significantly ameliorated the LPS-induced pathological alterations as demonstrated by the reduced cell infiltration and restored lobular architecture. Kidney tissues from ZIN-treated rats showed gradual but sustained recovery in cortex and medulla structure. Although the recovery is not full, it is easily noticeable in morphology. The thick descending and ascending parts of Henle loops and collecting coils of small caliber and reduction of interstitial tissue can be seen. The tubules show considerably lower cubic epithelium tubules were gaining normal morphology when treated with ZIN. The histopathological evaluation of lung samples indicated a moderate to severe hemorrhage, thickening of alveolar septa, emphysema, and infiltration of leukocytes in alveoli walls in the LPS group. Treatment with ZIN ameliorated these aberrations in the architecture of lung tissues. The improvement in histology is attributed to antioxidant and anti-inflammatory potential of ZIN as suggested by earlier reports [29,44]. Therefore, this research indicates that ZIN, in addition to its antioxidant and anti-inflammatory e ffects, may reduce the amount of circulating endotoxin from circulation and thereby alleviate the related multi-organ dysfunction syndrome (MODS) when administered to the animals subjected to LPS-induced sepsis.
