Ramadan A.M. Hemeida

Prophylactic role of curcumin in dextran sulfate sodium (DSS)-induced ulcerative colitis murine model.

We have addressed in this study the possible protective role of the main principle of turmeric pigment; curcumin on a murine model of ulcerative colitis (UC). Colitis was induced by administration of dextran sulfate sodium (DSS) (3% W/V) in drinking water to male Swiss albino rats for 5 consecutive days. DSS challenge induced UC model that was well characterized morphologically and biochemically. DSS produced shrinkage of colon length and increased the relative colon weight/length ratio accompanied by mucosal edema and bloody stool. Histologically, DSS produced submucosal erosions, ulceration, inflammatory cell infiltration and crypt abscess as well as epithelioglandular hyperplasia. The model was confirmed biochemically, and the test battery entailed elevated serum tumor necrosis factor (TNF-alpha) and colonic activity of myleoperoxidase (MPO). Colonic glutathione-S-transferase (GST) activity and its substrate concentration; GSH, were notably reduced, while lipid peroxidation, expressed as malondialdehyde (MDA) level, and total nitric oxide (NO) were significantly increased. Prior administration of curcumin (100mg/kg, IP) for 7 consecutive days ahead of DSS challenge mitigated the injurious effects of DSS and ameliorated all the altered biochemical parameters. These results suggest that curcumin could possibly have a protective role in ulcerative colitis probably via regulation of oxidant/anti-oxidant balance and modulation of the release of some inflammatory endocoids, namely TNF-alpha and NO.

Apoptotic DNA fragmentation may be a cooperative activity between caspase-activated deoxyribonuclease and the poly(ADP-ribose) polymerase-regulated DNAS1L3, an endoplasmic reticulum-localized endonuclease that translocates to the nucleus during apoptosis.

Background: The mechanism by which apoptotic internucleosomal DNA fragmentation occurs remains unclear. Results: CAD and DNAS1L3 cooperate to process chromatin degradation during apoptosis. DNAS1L3 achieves such function by translocating from the ER to the nucleus. Conclusion: The results provide new insight on the mechanism by which chromatin degradation takes place during apoptosis. Significance: Our results exemplify the complexity of chromatin degradation during apoptosis. Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca2+-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca2+ chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca2+ independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca2+ chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca2+-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.

Platelet‐derived growth factor‐BB induces cystathionine γ‐lyase expression in rat mesangial cells via a redox‐dependent mechanism

BACKGROUND AND PURPOSE So far, there is only limited information about the regulation of the endogenous synthesis of hydrogen sulfide (H2S), an important gaseous signalling molecule. This study was done to evaluate the redox‐dependent signalling events that regulate the expression of the H2S synthesising enzyme cystathionine‐γ‐lyase (CSE) in rat mesangial cells.

Acetyl-L-Carnitine Ameliorates Caerulein-induced Acute Pancreatitis in Rats

In the present study, we have addressed the possible protective role of acetyl-L-carnitine in caerulein-induced acute pancreatitis in male Swiss albino rats. Acute pancreatitis paradigm was developed by challenging animals with a supramaximal dose of caerulein (20 microg/kg, SC) four times at hourly intervals. Caerulein induced acute pancreatitis that was well-characterized morphologically and biochemically. Severe oedema with marked increased relative pancreatic weight, marked atrophy of acini with increased interacinar spaces, vacuolization, and extensive leucocytic infiltration were diagnostic fingerprints of the pancreatitis phenotype. A biochemical test battery that confirmed the model comprised increased plasma amylase and lipase activities, calcium levels as well as increased pancreatic enzymatic myeloperoxidase and glutathione-S-transferase activities, beside increased pancreatic contents of nitric oxide and malondialdehyde and reduced pancreatic glutathione level. Prior administration of acetyl-L-carnitine (200 mg/kg, IP) for seven consecutive days ahead of caerulein challenge alleviated all the histological and biochemical manifestations of acute pancreatitis. These results suggest a possible protective role of the carnitine ester in such a murine acute pancreatitis model probably via regulation of the oxidant/antioxidant balance, beside modulation of the myeloperoxidase and nitric oxide systems, which are involved in the inflammatory cascade that most often associate the disease.

ApoE deficiency promotes colon inflammation and enhances inflammatory potential oxidized-LDL and TNF-α in colon epithelial cells.

Although deficiency in Apolipoprotein E (ApoE) is linked to many diseases, its effect on colon homoeostasis remains unknown. ApoE appears to control inflammation by regulating nuclear factor-κB (NF-κB). The present study was designed to examine whether ApoE deficiency affects factors of colon integrity in vivo and given the likelihood that ApoE deficiency increases oxidized lipids and TNF-α, the present study also examined whether such deficiency enhances the inflammatory potential of oxidized-LDL (oxLDL) and TNF-α in colon epithelial cells (CECs), in vitro. Here we show that ApoE deficiency is associated with chronic inflammation systemically and in colonic tissues as assessed by TNF-α levels. Increased colon TNF-α mRNA coincided with a substantial increase in cyclooxygenase (COX)-2. ApoE deficiency enhanced the potential of oxLDL and TNF-α to induce COX-2 expression as well as several other inflammatory factors in primary CECs. Interestingly, oxLDL enhanced TGF-β expression only in ApoE−/−, but not in wild-type, epithelial cells. ApoE deficiency appears to promote COX-2 expression enhancement through a mechanism that involves persistent NF-κB nuclear localization and PI3 and p38 MAP kinases but independently of Src. In mice, ApoE deficiency promoted a moderate increase in crypt length, which was associated with opposing effects of an increase in cell proliferation and apoptosis at the bottom and top of the crypt respectively. Our results support the notion that ApoE plays a central role in colon homoeostasis and that ApoE deficiency may constitute a risk factor for colon pathologies.

Targeting Keap-1/Nrf-2 pathway and cytoglobin as a potential protective mechanism of diosmin and pentoxifylline against cholestatic liver cirrhosis

Aim: The effects of diosmin (DS), pentoxifylline (PTX) and their combination on inflammatory response, oxidant/antioxidant balance, cytoglobin and cirrhotic reaction during bile duct ligation (BDL) were investigated and explored. Main methods: Fifty adult male Wistar albino rats were randomly allocated to five groups as following, sham: received vehicle only, BDL: subjected to common BDL without treatment, BDL plus DS: received 100mg/kg/day orally, BDL plus PTX: received 50mg/kg/day orally, BDL plus DS plus PTX: received DS and PTX in the same manner. The test period lasted 28days, liver tissues and blood samples were collected to investigate biochemical markers (liver function biomarkers, oxidative stress markers, and antifibrotic markers), mRNA expression of Nrf‐2, Keap‐1, NF‐&kgr;B‐p65 and p38‐MAPK by real‐time PCR, protein expression of cytoglobin and NF‐&kgr;B‐p65 by western blot and iNOS and eNOS by immunohistochemistry. Histopathological study was performed to confirm our results. Key findings: Chronic BDL induced a significant alteration in liver functions, oxidative stress and fibrotic markers. Furthermore, unfavorable effects on gene and protein expression were observed after BDL. Histopathological findings of this group showed parallel effects. DS, PTX and their combination treatment significantly ameliorated the disturbance that occurred due to BDL. Similar findings were observed in liver histopathology. Significance: DS and PTX could mitigate liver cirrhosis through modulation of Keap‐1/Nrf‐2/GSH and NF‐&kgr;B‐p65/p38‐MAPK signaling pathways. In addition, we demonstrated that the hepatoprotective effect of DS and PTX is mediated by up‐regulation of cytoglobin with inhibition of fibrotic reaction.

Hepatoprotective effects of diosmin and/or sildenafil against cholestatic liver cirrhosis: The role of Keap-1/Nrf-2 and P38-MAPK/NF-κB/iNOS signaling pathway

The present study was designed to investigate the potential protective effects of diosmin (DS) and/or sildenafil against bile duct ligation (BDL). In order to achieve this goal, BDL was performed to induce liver cirrhosis, DS (100 mg/kg/day, p.o.) and sildenafil (10 mg/kg, twice daily, p.o.) were administrated alone or in combination 24 h after the surgical operation and lasted for 4 weeks. Liver function biomarkers, fibrotic markers, oxidative stress markers, mRNA expression of NF-κB-p65, P38-MAPK, Nrf-2, and Keap-1, as well as protein expression of cytoglobin, NF-κB-p65, Nrf-2, iNOS and eNOS were investigated concomitantly with histopathological study. The results revealed that, 4 weeks of BDL induced a significant alteration in liver functions, fibrotic and oxidative stress markers. Furthermore, up-regulation of NF-κB-p65, P38-MAPK, Keap-1 and iNOS concomitantly with down-regulation of Nrf-2, cytoglobin and eNOS expressions were observed after BDL. DS and/or sildenafil treatment significantly alleviated the disturbance induced by BDL. These findings were further supported by the improvement in histopathological features. Additionally, co-administration of DS and sildenafil were found to significantly improved liver defects due to BDL as compared to the individual drugs. It can be concluded that, DS and sildenafil exhibit hepatoprotective effects through modulation of Keap-1/Nrf-2 and P38-MAPK/NF-κB/iNOS pathway.

Perindopril ameliorates lipopolysaccharide-induced brain injury through modulation of angiotensin-II/angiotensin-1-7 and related signaling pathways

ABSTRACT Localized tissue renin‐angiotensin system (RAS) is an interesting pathway of organ damage. Here, the effect of the brain‐penetrating angiotensin converting enzyme (ACE) inhibitor perindopril was studied on lipopolysaccharide (LPS)‐induced brain damage, with and without exogenous angiotensin (Ang)‐II administration. Animals were divided into 6 groups; a normal control group, an LPS control group (LPS, 3mg/kg, i.p., single dose), two treatment groups receiving perindopril (1 and 2mg/kg/day, i.p.) for 7 days before LPS administration, and two Ang‐II/perindopril/LPS groups receiving perindopril and LPS, followed by a single dose of Ang‐II solution (5 &mgr;l, i.c.v.). Brain tissue Ang‐II, Ang‐1–7, and NADPH oxidase were estimated using ELISA technique. Nuclear factor kappa‐B (NF‐&kgr;B‐p65) was estimated using real time PCR technique, while phosphorylated NF‐&kgr;B‐p65 (p‐NF‐&kgr;B‐p65), phosphorylated and non‐phosphorylated protein kinase B (p‐Akt and Akt) and phosphorylated inhibitor of kappa‐B (p‐I&kgr;Ba) were estimated by western blot analysis. Malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione‐S‐transferase (GST), catalase, nitrite and myloperoxidase (MPO) were estimated colorimetrically. Brain tissue inducible and endothelial nitric oxide synthases (iNOS and eNOS) were estimated immunohistochemically, confirmed by a histopathological study. LPS‐intoxicated rats showed significantly elevated Ang‐II, NADPH oxidase, NF‐&kgr;B‐p65, p‐NF‐&kgr;B‐p65, p‐I&kgr;Ba, p‐Akt, Akt, p‐Akt/Akt ratio, MDA, nitrite, MPO and iNOS levels, coupled with significantly suppressed Ang‐1–7, GSH, SOD, GST, catalase, and eNOS levels, which were all corrected by pre‐treatment with perindopril in both doses by varying degrees. Exogenous Ang‐II significantly ameliorated the protective effects of perindopril. Conclusively, perindopril ameliorates LPS‐induced brain damage through modulation of RAS, iNOS/eNOS, p‐Akt/Akt and NF‐&kgr;B signaling pathways.

Abstract 4171: Lipid metabolism-independent role of apolipoprotein (E) levels in colon carcinogenesis through a regulating inflammation and active β-catenin

Apolipoprotein E (ApoE) is critically important for cholesterol metabolism and lipid homeostasis. Polymorphisms in the ApoE gene have been implicated in the development of colon cancer and these polymorphisms may be used as risk indicators for the disease. Accruing evidence suggests new lipid metabolism-independent functions for ApoE both in inflammation and cardiovascular diseases. Ironically, ApoE was shown to up-regulate cyclooxygenase-2 in vascular smooth muscle cells and given the critical role of the COX-2 in colon carcinogenesis, these results suggest a pro-colon cancer function for the lipoprotein. In the present study, we show that a mere deletion of ApoE promotes systemic inflammation as assessed by TNF-α level in sera of mice under a regular diet. Concomitantly, TNF-α mRNA levels were significantly higher in the colon of ApoE-/- mice suggestive of localized inflammation. In contrast to smooth muscle cells, in vitro studies with primary colon epithelial cells (CECs) show that ApoE-deficiency substantially increased COX-2 expression in response to oxidized (ox)LDL both at the protein and mRNA levels. OxLDL-induced expression of COX-2 in WT CECs was highly sensitive to PP2 treatment, a Src inhibitor, and to LY294002, a pan-PI3K inhibitor. Surprisingly, while oxLDL-induced COX-2 expression was sensitive to PI3K inhibition, its sensitivity to Src inhibition was minimal suggesting that ApoE deficiency may promote COX-2 expression through a Src-independent mechanism. Similarly to COX-2, ApoE-deficiency also enhanced basal and ox-LDL-induced expression of MCP-1, IL-1β, ICAM-1, and VCAM-1. Using a cohort of colon cancer patients and healthy controls, we show that the overall levels of ApoE in sera of colon cancer patients were significantly lower than those of healthy controls (p = 0.0028). We have utilized ApcMin mice, well-characterized mouse model of intestinal tumor. Using this genetic background, we have generated ApoE+/- and ApoE-/- lines which were utilized in this study. We found that inhibition of ApoE through gene heterozygosity led to ∼50% reduction in the level of ApoE protein and a significant increased the number tumors in these mice compared to their wildtype counterparts. These changes in tumor burden were accompanied by slight changes in the lipid profiles of the ApoE+/- mice. Surprisingly however, when examining the results of ApoE-/-, we found that the tumor burden was not increased over that seen in the ApoE heterozygous mice, but the lipid profiles were elevated dramatically. This suggests that the mechanism through which ApoE affects lipid metabolism is separate from that which is involved in the formation of colon tumors. This aggravation of the tumor burden may be associated with a stimulus-independent increase in the levels of the active form of β-catenin. These results suggest that ApoE deficiency may be informative for the risk of developing colon cancer. Citation Format: Abdelmetalab F. Tarhuni, Ali H. El-Bahrawy, Hogyoung Kim, Youssef Errami, Mohamed A. Ghonim, Mohamed Alwan, Samar M. Hammad, Venkat N. Subramaniama, Ramadan A. Hemeida, Amarjit S. Naura, Hamid A. Boulares. Lipid metabolism-independent role of apolipoprotein (E) levels in colon carcinogenesis through a regulating inflammation and active β-catenin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4171.

Abstract LB-3: Apolipoprotein (E) is a determinant of colon carcinogenesis potentially by regulating inflammation and β-catenin independently of its role in lipid metabolism

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Colorectal cancer is a leading cause of cancer-related mortality. Apolipoprotein E (ApoE) is a major player in cholesterol metabolism and has been suggested, through association studies, to play a role in colon homoeostasis and cancer. Genetic studies suggested that polymorphisms in the ApoE alleles constitute a risk factor for the development of colon cancer. Collectively, these studies explored the role of ApoE in colon cancer by narrowly focusing on its established function in lipid metabolism. In the current study, we utilized a genetic mouse model (ApcMin mouse) of colon cancer to test the hypothesis that ApoE is a determinant in the disease progression. We show that ApoE gene heterozygosity, which reduces the protein (∼50%) and minimally affects the lipid profile, significantly increased tumor burden in ApcMin mice both in the small intestine and the colon. Surprisingly, ApoE gene knockout, which completely eliminates the protein and drastically elevates the lipid profile, did not aggravate the tumor burden in ApcMin mice compared to that caused by gene heterozygosity, suggesting a novel and cholesterol-metabolism-independent function for ApoE in colon homeostasis. In vitro studies with primary colon epithelial cells, isolated using a novel technique developed by our laboratory, show that ApoE-deficiency achieved by ApoE heterozygosity, knockout, or knockdown increased the level of active β-catenin in the absence of any stimulation. It appears that such increase in β-catenin may be at an mRNA level. ApoE deficiency was also associated with an increase in inflammatory factors including VCAM-1, MCP-1, and COX-2 in TNF-treated colon epithelial cells. Altogether, our preliminary results suggest a major role of ApoE in colon homeostasis and that deficiency in ApoE levels may constitute a risk factor for colon cancer progression. Citation Format: Ali I. Elbahrawy, Abdelmetalab Tarhuni, Amarjit S. Naura, Youssef Errami, Mohamed Alwan, Samar Hammad, Jimena Trillo-Tinoco, Ramadan Hemeida, Hassan Brim, Hassan Ashktorab, Luis Del Valle, Hamid A. Boulares. Apolipoprotein (E) is a determinant of colon carcinogenesis potentially by regulating inflammation and β-catenin independently of its role in lipid metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-3. doi:10.1158/1538-7445.AM2014-LB-3