الفهرس 
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Abstract
Summary
7.1. Aim of work:
This study aimed to investigate the protective effects of H. salicornicum ethanolic and methanolic extracts against CIS-induced hepato- and nephrotoxicity with an emphasis on their modulatory effect on oxidative stress, inflammation, and apoptosis. The role of Nrf2/HO-1 signaling and micro-RNA-34a/SIRT1signaling in mediating the protective effects of H. salicornicum extracts against CIS toxicity was evaluated. Phytochemical analysis of the ethanolic and methanolic extracts of H. salicornicum was conducted.
7.2. Materials & Methods:
To investigate the protective effect of HSE (ethanolic extract) on nephrotoxicity of CIS, 48 the rats were divided into six groups (n=6) as follows:
group I (Control): received 0.5% carboxymethyl cellulose (CMC) for 14 days via oral gavage and a single intraperitoneal (i.p.) injection of physiological saline at day 7.
group II (HSE): rats received 400mg/kg of body weight HSE dissolved in 0.5% CMC for 14 days and a single i.p. injection of physiological saline at day 7.
group III (CIS): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 0.5% CMC via oral gavage for 14 days.
group IV (CIS + 100 mg/kg HSE): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 100mg/kg body weight HSE dissolved in 0.5% CMC for 14 days.
group V (CIS + 200 mg/kg HSE): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 200mg/kg body weight HSE dissolved in 0.5% CMC for 14 days.
group VI (CIS + 400 mg/kg HSE): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 400mg/kg body weight HSE dissolved in 0.5% CMC for 14 days.
To investigate the protective effect of HSE (methanolic extract) on hepatotoxicity of CIS, 48 the rats were divided into six groups (n=6) as follows:
group I (Control): received 0.5% carboxymethyl cellulose (CMC) for 14 days via oral gavage and a single intraperitoneal (i.p.) injection of physiological saline at day 7.
group II (HSE): rats received 400mg/kg of body weight HSE dissolved in 0.5% CMC for 14 days and a single i.p. injection of physiological saline at day 7.
group III (CIS): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 0.5% CMC via oral gavage for 14 days.
group IV (CIS + 100 mg/kg HSE): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 100mg/kg body weight HSE dissolved in 0.5% CMC for 14 days.
group V (CIS + 200 mg/kg HSE): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 200mg/kg body weight HSE dissolved in 0.5% CMC for 14 days.
group VI (CIS + 400 mg/kg HSE): rats received a single i.p. injection of CIS (7mg/kg body weight) at day 7 and 400mg/kg body weight HSE dissolved in 0.5% CMC for 14 days.
7.3. Results:
Nephrotoxicity was seen in 28–36% of patients received CIS alone at dose of 50 mg/m2. Within 2 to 6 days after the administration of CIS, acute oliguric or non-oliguric renal insufficiency occur. uric acid in the circulation and/or a decrease in creatinine clearance and imbalanced electrolytes. A one possible way to reduce the risk of CIS nephrotoxicity is the aggressive hydration of at least 3–6 L per day which decrease the reactive monohydrated cisplatin form. Serum creatinine, urea and Kim-1 were assayed to evaluate the protective effect of HSE on CIS nephrotoxicity. CIS administration resulted
in a significant elevation of creatinine, urea and Kim-1 when compared with the control rats (P<0.001). Treatment with 100, 200 or 400 mg/kg HSE ameliorated serum creatinine, urea, and Kim-1 levels in CIS-intoxicated rats. The effect of HSE on the assayed kidney function markers was dose dependent. Oral supplementation of 400 mg/kg HSE did not alter liver function in normal rats.
Microscopic examination revealed normal structure of the kidney in control rats. Rats in this group exhibited normal glomeruli, renal tubules and Bowman’s capsules and showed no pathologic alterations. CIS administration caused several histopathological alterations, including glomerular atrophy, degenerative changes, desquamation of the tubular epithelia, and deformity of some glomeruli with widening of glomerular space. Treatment with HSE at doses of 100, 200 and 400 mg/kg resulted in a remarkable improvement of the histological architecture of the kidney of rats.
the ameliorative effect of HSE on CIS-induced redox imbalance in the kidney of rats. ROS levels exhibited a significant elevation in the kidney of CIS-intoxicated rats when compared with the control group (P<0.001) as illustrated. Oral supplementation of HSE decreased renal ROS in CIS-treated rats in a dose-dependent manner. Renal MDA, and NO were significantly increased in CIS-intoxicated rats. These alterations were reversed in rats treated with 100, 200 and 400 mg/kg HSE. In contrast, CIS decreased renal GSH content and the activity of SOD and CAT significantly when compared with the control group. All doses of HSE increased renal GSH and antioxidant enzymes in CIS-intoxicated rats. Of note, the 400 mg/kg dose of HSE had no effect on ROS, MDA, NO and antioxidant defenses in the kidney of normal rats.
Changes in the expression of NF-κB p65, iNOS, TNF-α and IL-1β were determined to explore the ameliorative effect of HSE on CIS-induced inflammatory response in the kidney of rats. The results revealed a significant upregulation of NF-κB p65 in the kidney of CIS-intoxicated rats (P<0.001) when compared with the control group . HSE showed a
potent suppressive effect where all doses downregulated renal NF-κB p65 markedly in the kidney of CIS-intoxicated rats.
Similar to NF-κB p65, TNF-α , IL-1β and iNOS were significantly upregulated in the liver kidney of CIS-intoxicated rats when compared with the control group (P<0.001). HSE remarkably ameliorated renal iNOS, TNF-α and IL-1β expression in rats that received CIS (P<0.001). Of note, HSE exerted a dose-dependent ameliorative effect on renal iNOS, TNF-α and IL-1β in CIS-intoxicated rats, whereas had no effect in normal rats.
Molecular docking simulations revealed the binding affinity of the isolated compounds (1-7) with NF-κB. Compounds 7 (isorhamnetin-3-O-β-D-glucopyranoside), 5 (hyperoxide) and 2 (myricetin) showed the highest binding affinity with docking scores -10.7, -10.2 and -9.7 (kal/mole), respectively. Isorhamnetin-3-O-β-D-glucopyranoside forms polar bonds with the residues Lys218, Arg246 and Arg305 and exhibits hydrophobic interactions Arg33, Asn186, Arg187, Asp217, Gln247, Val248, Gln306 and Phe3 The mRNA abundance of BCL-2 and BAX, caspase-3 and immunostaining of caspase-3 were determined to evaluate the protective effect of HSE on CIS-induced apoptosis in the kidney of rats. CIS increased BAX and casepase-3 and decreased BCL-2 mRNA significantly (P<0.001) in the kidney of rats. In addition, CIS-intoxicated rats exhibited a significant increase in renal caspase-3 immunostaining when compared with the control rats (P<0.001). HSE upregulated renal BCL-2 and decreased BAX and caspase-3 rats received CIS. The effect of HSE on BAX and caspase-3 was dose dependent in CIS-intoxicated rats and the highest dose had no effect in normal rats.
Keap1 mRNA abundance exhibited a significant increase and Nrf2 and HO-1 mRNA were decreased in the kidney of CIS-intoxicated rats when compared with the control rats (P<0.001). HO-1 activity was also decreased in the kidney of rats that received CIS. Treatment with HSE (100, 200 and 400 mg/kg) remarkably ameliorated renal Keap1, Nrf2 and HO-1 expression and HO-1 activity in CIS-intoxicated rats.
qPCR was employed to evaluate changes microRNA-34a and Sirt1in the kidney of CIS-intoxicated rats treated with HSE. In addition, we conducted a molecular docking study to investigate the binding affinity of the isolated compounds with Sirt1. The results revealed a significant upregulation of microRNA-34a and Sirt1 mRNA in the kidney of CIS-intoxicated rats when compared with control group (P<0.001). HSE (100, 200 and 400 mg/kg) significantly ameliorated renal microRNA-34a and Sirt1levels in CIS-intoxicated rats. In addition, HSE increased Sirt1 protein expression in the kidney of CIS-intoxicated.
CIS induced hepatotoxicity has been demonstrated to be a result of oxidative stress and manifested by elevated serum transaminases and bilirubin. After the administration of CIS, glutathione (GSH) content and glutathione reductase activity were decreased, whereas GSH peroxidase, catalase (CAT) and gamma-glutamyl transpeptidase were increase. The information on Cisplatin-induced liver injury and its mechanism in causing hepatotoxicity is very less. It is known that the drug accumulates in significant amount in hepatic tissue, particularly when injected in high doses. CIS administration can enhance cytochrome-P450-2E1 and subsequently provoke liver injury. Owing to the role of oxidative stress in mediating CIS hepatotoxicity, the use of high doses of antioxidants such as vitamin E and selenium ameliorated the toxic effect of CIS on the liver.
Serum ALT, AST and ALP were assayed to evaluate the protective effect of HSE on CIS hepatotoxicity (Table 5.1). CIS administration resulted in a significant elevation of ALT (Fig. 5.2A), AST (Fig. 5.2B) and ALP (Fig. 5.2C) when compared with the control rats (P<0.001). Treatment with 100, 200 or 400 mg/kg HSE ameliorated serum ALT, AST and ALP in CIS-intoxicated rats. The effect of HSE on the assayed liver function markers was dose dependent. Oral supplementation of 400 mg/kg HSE did not alter liver function in normal rats.
CIS-induced rats revealed disrupted organization of hepatic cords, congested blood vessels, inflammatory cell infiltration, necrosis, vacuolar and hydropic degeneration in hepatocytes.
treatment with 100, 200 and 400 mg/kg HSE ameliorated CIS-induced liver injury and improved the histological architecture where only slight congestion could be seen.
ROS levels exhibited a significant elevation in the liver of CIS-intoxicated rats when compared with the control group (P<0.001). Oral supplementation of HSE decreased hepatic ROS in CIS-treated rats in a dose-dependent manner. Hepatic MDA, a LPO marker and NO were significantly increased in CIS-intoxicated rats. These alterations were reversed in rats treated with 100, 200 and 400 mg/kg HSE. Changes in the expression of NF-κB p65, iNOS, TNF-α and IL-1β were determined to explore the ameliorative effect of HSE on CIS-induced inflammatory response in the liver of rats. Immunohistochemical investigation revealed a significant upregulation of NF-κB p65 in the liver of CIS-intoxicated rats (P<0.001). HSE showed a potent suppressive effect where all doses downregulated hepatic NF-κB p65 markedly in CIS-intoxicated rats. These findings were supported by ELISA results that revealed a significant decrease in hepatic NF-κB p65 in CIS-administered rats treated with HSE.
Similar to NF-κB p65, iNOS, TNF-α and IL-1β were significantly upregulated in the liver of CIS-intoxicated rats when compared with the control group (P<0.001). HSE remarkably ameliorated hepatic iNOS, TNF-α and IL-1β expression in rats that received CIS (P<0.001). Of note, HSE exerted a dose-dependent ameliorative effect on hepatic NF-κB p65, iNOS, TNF-α and IL-1β in CIS-intoxicated rats, whereas had no effect in normal rats. The mRNA abundance of BCL-2 and BAX, immunostaining of caspase-3 and 8-Oxo-dG were determined to assess the beneficial effect of HSE against CIS-induced apoptosis and oxidative DNA damage in the liver of rats. CIS downregulated BCL-2 mRNA whereas BAX mRNA and BAX/BCL-2 ratio was increased significantly (P<0.001) in the liver of rats. In addition, CIS-intoxicated rats exhibited a significant increase in hepatic caspase-3 immunostaining and 8-Oxo-dG levels when compared with the control rats (P<0.001). HSE upregulated hepatic BCL-2 and decreased BAX, BAX/BCL-2 ratio, caspase-3 and 8-Oxo-dG in rats received CIS. The effect of HSE on BAX, caspase-3 and 8-Oxo-dG was dose dependent in CIS-intoxicated rats and the highest dose had no effect in normal rats. Nrf2 mRNA abundance exhibited a significant decrease in the liver of CIS-intoxicated rats
when compared with the control rats (P<0.001). HO-1 activity was also decreased in the liver of rats that received CIS. Treatment with HSE (100, 200 and 400 mg/kg) remarkable ameliorated hepatic Nrf2 expression and HO-1 activity in CIS-intoxicated rats.
qPCR was employed to evaluate changes microRNA-34a and Sirt1in CIS-intoxicated rats treated with HSE. In addition, we conducted a molecular docking study to investigate the binding affinity of the isolated compounds with Sirt1. The results revealed a significant upregulation of microRNA-34a and Sirt1 mRNA in the liver of CIS-intoxicated rats when compared with control group (P<0.001). HSE (100, 200 and 400 mg/kg) significantly ameliorated hepatic microRNA-34a and Sirt1levels in CIS-intoxicated rats.
7.4. Conclusions:
These findings provide new information on the protective effect of H. salicornicum against CIS hepato-and nephrotoxicity via their dual ability to attenuate oxidative stress , inflammation and programmed cell death. prevented surplus production of ROS, inflammation and apoptosis, and enhanced antioxidant defenses by activating Nrf2/HO-1 signaling and micro-RNA-34a/SIRT1signaling in mediating the protective effects of H. salicornicum extracts against CIS toxicity was evaluated. Phytochemical analysis of the ethanolic and methanolic extracts of H. salicornicum was conducted. HSE can activate NF-κB/NLRP3 inflammasome axis and its down-stream molecules caspase-1 and IL-1β in the kidney of rats. This investigation provides new information on the potential of HSE to mitigate CIS nephrotoxicity. HSE markedly prevented overproduction of ROS, inflammation and apoptosis, and enhanced antioxidant defenses in rat kidney. Activation of Nrf2/HO-1 signaling and micro-RNA-34a/SIRT1signaling in mediating the protective effects of H. salicornicum extracts against CIS toxicity was evaluated. Phytochemical analysis of the ethanolic and methanolic extracts of H. salicornicum was conducted. suppression of ROS and NF-κB/NLRP3 inflammasome signaling are the main mechanisms
underlying the ameliorative effect of HSE. Therefore, HSE could be used as an adjuvant therapy or a supplement to protect against hepatotoxicity and AKI in patients who receive CIS, pending further studies to assess its exact mechanisms of action.