**2. Results**

#### *2.1. E*ff*ect of ZIN on Survival Rate of Animals*

The survival rate of animalswasmonitored up to 96 h andisillustratedin Figure 2. Zingerone treatment increases the animal survival rate (70%) in lipopolysaccharide (LPS)-challenged animals.

**Figure 2.** Kaplan–Meier survival plot (n = 10 rats per group). Effect of zingerone (ZIN) 150 mg/kg on survival rate of LPS induce systemic inflammation in rats.

#### *2.2. E*ff*ect of ZIN on Biochemical Markers*

Zingerone treatment exhibit minor changes in the biochemical parameters in the rats as compared with normal rats (Table 1). Animals in the endotoxemia (LPS) group showed significant abnormalities in different organ function markers as apparent by enhanced biochemical marker levels. The hepatic injury markers ALT (145.02 ± 3.84, U/L), ALP (186.54 ± 8.76, U/L) and AST (206.49 ± 11.45, U/L) levels were significantly higher in LPS group as compared to the control group ALT (27.69 ± 3.20, U/L), ALP (64.24 ± 4.32, U/L), and AST (57.65 ± 3.76, U/L), respectively. ZIN treatment decreases hepatic marker levels significantly from ALT (145.02 ± 3.84 to 74.99 ± 6.69 U/L, *p* < 0.05), ALP (186.54 ± 8.76 to 91.29 ± 5.34 U/L, *p* < 0.05) and AST (206.49 ± 11.45 to 87.35 ± 8.29 U/L, *p* < 0.05). General tissue damage of rats was confirmed from serum LDH levels in LPS group, a significantly increase in LDH levels were observed (972.75 ± 20.74 U/L), and these levels were decreased with ZIN treatment (656.59 ± 7.46 U/L *p* < 0.05). Similarly, a reduction in CK (553.83 ± 19.61 U/L *p* < 0.05), SCr (0.38 ± 0.05 mg/dL *p* < 0.05) and, BUN (67.71 ± 2.27 mg/dL *p* < 0.05) were observed in ZIN treated groups as compared to the animals in LPS group CK (855.47 ± 18.84 U/L), SCr (0.59 ± 0.09 mg/dL), and BUN (153.18 ± 7.34 mg/dL), respectively. Also, albumin levels were also increased with the ZIN treatment (3.18 ± 0.013 g/dL *p* < 0.05) as compared to the animals in the LPS group (2.36 ± 0.12 g/dL).


**Table 1.** List of plasma biochemical levels of different groups.

Results are represented as mean ± SEM of six rats/group. *\* p* < 0.05 vs. control; # *p* < 0.05 vs. LPS group. BUN: Blood urea Nitrogen; LDH: Lactate dehydrogenase; ALT: Alanine transaminase; ALP: Alkaline phosphatase; AST: Aspartate transaminase; BIL: Bilirubin; GGT: Gamma-glutamyl transferase.

#### *2.3. E*ff*ect of ZIN on Oxidative Stress and Antioxidant Enzyme Markers*

The LPS injection elicited levels of NO (97.08 ± 3.64 μM), which was significantly diminished in ZIN treated animals (57.36 ± 3.50 μM *p* < 0.05). 8-OHdG an indicator of DNA damage, the levels were significantly enhanced in the LPS treated groups (10.02 ± 0.84 ng/mL) and this was decreased with ZIN treatment (5.26 ± 0.40 ng/mL *p* < 0.05), respectively (Figure 3). Oxidative stress markers in different tissues e.g., brain, kidney, lung, and liver were measured in terms of glutathione (GSH), malondialdehyde (MDA), and myeloperoxidase (MPO) levels (Figures 4–6). MPO an enzyme of activated Polymorphonuclear (PMN) were used as an indication of tissue neutrophil accumulation. ZIN treatment in all tissues showed a significant (*p* < 0.05) reduction of these oxidative markers which was increased in LPS treated animals.

**Figure 3.** Effect of zingerone (ZIN) on LPS-induced DNA damage and oxidative stress marker. (**A**) 8-OHdG levels (ng/mL), (**B**) Nitric Oxide (NO) levels (μM). Results are represented as mean ± SEM of six rats/group. \* *p* < 0.05 vs. control; # *p* < 0.05 vs. LPS group.

**Figure 4.** Effect of zingerone (ZIN) on LPS-induced oxidative stress as malondialdehyde (MDA) (nmol/kg). (**A**) Brain, (**B**) lung, (**C**) liver, and (**D**) kidney. Results are represented as mean ± SEM of six rats/group. \* *p* < 0.05 vs. control; # *p* < 0.05 vs. LPS group.

**Figure 5.** Effect of zingerone (ZIN) on LPS-induced oxidative stress as GSH (μmol/kg). (**A**) Brain, (**B**) Lung, (**C**) Liver and (**D**) Kidney. Results are represented as mean ± SEM of six rats/group. \* *p* < 0.05 vs. control; # *p* < 0.05 vs. LPS group.

**Figure 6.** Effect of zingerone (ZIN) on LPS-induced oxidative stress as MPO (U/g). (**A**) Brain, (**B**) lung, (**C**) liver, and (**D**) kidney. Results are represented as mean ± SEM of six rats/group. \* *p* < 0.05 vs. control; # *p* < 0.05 vs. LPS group.

#### *2.4. E*ff*ect of ZIN on Cytokines and Inflammatory Markers*

The inflammatory changes in the LPS-induced animals were determining as their cytokine levels (Figure 7). LPS-induced rats demonstrated a significant increase in the plasma levels of TNF-α (135.56 ± 4.17 pg/mL), IL-1α (509.12 ± 17.79 pg/mL), IL-1ß (1166.01 ± 27.54 pg/mL), IL-2 (20.67 ± 1.70 pg/mL), IL-6 (106.56 ± 3.44 pg/mL) and IL-10 (1037.49 ± 31.65 pg/mL). Zingerone treated animals showed reduced levels of these cytokines as compared to the disease control animals TNF-α (60.16 ± 3.52 pg/mL *p* < 0.05), IL-1α (235.49 ± 10.07 pg/mL *p* < 0.05), IL-1ß (739.78 ± 25.57 pg/mL *p* < 0.05), IL-2 (14.19 ± 1.77 pg/mL *p* < 0.05), IL-6 (61.60 ± 3.21 pg/mL *p* < 0.05) and IL-10 (665.90 ± 14.17 pg/mL *p* < 0.05). PCT levels were determined in the plasma of rats and were significantly increased in the LPS treated animals (2351.65 ± 39.75 pg/mL *p* < 0.05) compared to the normal rats (1053.03 ± 49.49 pg/mL). ZIN treatment significantly diminish the plasma PCT levels (1626.83 ± 86.70 pg/mL *p* < 0.05) Figure 7G.

#### *2.5. E*ff*ect of ZIN on Histoarchitecture of Di*ff*erent Organs*

As depicted in Figure 8, the tissue sections from normal control group of different organs (brain, lung, liver, and kidney) exhibit normal architecture. Brain, lung, liver, and kidney tissues from LPS group exhibited the pathological alterations comprising widespread inflammation, portal inflammation, and hepatic cell necrosis, infiltration of inflammatory cells, severe hemorrhage, and thickening of alveolar septa, emphysema, and infiltration of leukocytes in walls alveoli and neuronal loss and condensed nucleus. However, treatment with ZIN 150 mg/kg significantly prevented the LPS-induced pathological changes and restored the histological architecture.

**Figure 8.** Light histograms of different rat organs (hematoxylin and eosin stains, magnification 40× and scale bar of 100 μm). Effect of ZIN in LPS intoxicated rats. Brain (**A**) normal control group: Showing normal histological structure with intact neurons, (**B**) LPS treated rats: Showing neuronal loss with condensed nuclei, (**C**) Rats treated with (ZIN 150 mg/kg + LPS: Showing decrease in neuronal loss with presence of intact neurons. Kidney (**A**) normal control group: Showing normal histological structure of the glomeruli and tubules at the cortex with absence of histopathological alterations, (**B**) LPS treated rats: Showing marked inflammatory cell aggregation in between the tubules, marked degeneration in the lining epithelium of all the tubules, and blood vessel congestion, (**C**) Rats treated with (ZIN 150 mg/kg + LPS: Showing absence of histopathological alterations. Liver (**A**) Normal control group: Showing normal histological structure of the central vein and intact hepatocytes, (**B**) LPS treated rats: Showing severe loss of hepatic architecture with multiple focal necrosis, ballooning degeneration in the hepatocytes, (**C**) Rats treated with ZIN 150 mg/kg + LPS: Showing absence of histopathological alterations. Lung (**A**) Normal control group showing normal morphology, (**B**) LPS treated rats: Showing moderate to severe hemorrhage, thickening of alveolar septa, emphysema, and infiltration of leukocytes in walls alveoli, (**C**) Rats treated with ZIN 150 mg/kg + LPS: Showing absence of histopathological changes.
