الفهرس 
Overview on Ischemia-ReperfusionOnly 14 pages are availabe for public view
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Abstract
The present study was planned to determine the potential protective effect of bone marrow-derived mesenchymal stem cells alone and/or in combination with vitamin D against hepatic ischemia/reperfusion injury induced in rats and to explore the possible underlying mechanisms.
The study was performed on 60 adult male Wistar rats, which were allocated into 5 groups:
group I: Sham-operated control group (Sham) (n=12):
Rats in this group were subjected to oral intake of distilled water (D.W.) in a volume of 0.05 ml by gavage, 5 days/week for 2 weeks, followed by a single intravenous (IV) injection of 0.5 ml phosphate buffer saline (PBS) in rat tail vein half an hour prior to laparotomy and closure without vascular clamping.
group II: Hepatic Ischemia-Reperfusion group (IR) (n=12):
Rats in this group were subjected to oral intake of D.W. in a volume of 0.05 ml by gavage, 5 days/week for 2 weeks, followed by a single intravenous (IV) injection of 0.5 ml phosphate buffer saline (PBS) in rat tail vein half an hour prior to 1-hour partial liver ischemia followed by 24-hours reperfusion according to Bejaoui et al. (2016) & Liu et al. (2016).
group III: Vitamin D treated, Hepatic IR group (Vit D-IR) (n=12):
Rats in this group were subjected to oral intake of Vitamin D3 by gavage, in a dose of 500 IU/Kg daily (Chabas et al., 2013), 5 days/week for 2 weeks, followed by a single IV injection of 0.5 ml PBS in rat tail vein half an hour prior to hepatic IR as in group II.
group IV: Stem cell treated, Hepatic IR group (Stem cell-IR) (n=12):
Rats in this group were subjected to oral intake of D.W. as in group I, followed by a single IV injection of 1×106 bone marrow-derived mesenchymal stem cells (BM-MSCs) within 0.5 ml PBS in rat tail vein half an hour prior to hepatic IR (Wang et al., 2018).
group V: Stem cell and Vitamin D treated, Hepatic IR group (Combined-IR) (n=12):
Rats in this group received Vitamin D3 as in group III and BM-MSC as in group IV before being subjected to hepatic IR as group II.
All rats were subjected to assessment of body weight (BW), absolute liver weight (LW) for calculation of liver index (LI) as well as the measurement of the serum levels of alanine and aspartate aminotransferase (ALT, AST) and serum Vitamin D (Vit D) levels, and determination of oxidative stress by assessing the hepatic tissue level of Malondialdehyde (H.MDA) and superoxide dismutase (H.SOD), the inflammatory response by evaluating the level of interleukin-2 (H.IL-2) as an inflammatory marker and interleukin-10 (H.IL-10) as an anti-inflammatory marker, autophagy by measuring hepatic tissue level of LC3B (H.LC3B), apoptosis by assessing hepatic tissue level of Caspase-3 (H.Casp-3), in addition to the regenerative capacity by hepatic tissue level of hepatocyte growth factor (H.HGF), beside histopathological liver examination and scoring. Fluorescence imaging of labeled BM-MSCs was also done for the Stem cell-IR and Combined-IR groups.
The results of the present study can be summarized as follows:
Rats that underwent IR without pretreatment showed significant decreases in LW, LI, and serum levels of Vit. D, hepatic tissue levels of H.SOD, H.IL-10, H.LC3B, and H.HGF. These changes were accompanied by significant increases in serum levels of ALT and AST and hepatic tissue levels of H.MDA, H.IL-2, and H. Casp-3 as compared to the Sham group. In addition to marked sinusoidal congestion, cytoplasmic vacuolization and cellular necrosis as shown in the histopathological examination with the worst score among all studied groups.
Upon pretreatment with Vitamin D in the Vit D-IR group, there was still a significant decrease in LW, and LI compared to the sham group. However, significant reductions were observed in the serum levels of ALT and AST and hepatic tissue levels of H.MDA, H.IL-2, and H. Casp-3, whereas serum levels of Vit. D and hepatic levels of H.SOD, H.IL-10, H.LC3B, and H.HGF showed significant increases compared to the IR group, yet still significantly different from the Sham group. In addition, there was a partial improvement in the histopathological study with the worst score among the treated groups.
Regarding the Stem cell pretreated group, LW, and LI showed significant increases compared to the IR group being comparable from Sham regarding LI only, yet LW was still significantly less than Sham. Furthermore, serum levels of both ALT and AST and hepatic tissue levels of H.MDA, H.IL-2, and H. Casp-3 were significantly decreased when compared to the IR group, yet still significantly higher than the Sham group. Hepatic tissue levels of H.SOD, H.IL-10, H.LC3B, and H.HGF were significantly increased compared to the IR group but still significantly less than the Sham group. Compared to the Vit D-IR group, AST was significantly higher. Serum levels of Vit. D showed a significant increase as compared to the IR group, but a significant decrease as compared to Vit. D-IR group, being comparable from the Sham group. In addition to the partial improvement in the histopathological studies and grading.
In rats that received the combined treatment, there was a significant increase in LW compared to IR and Vit D-IR groups, yet still significantly less than Sham. LI showed a significant increase compared to the IR group being comparable to Sham. Compared to the IR, Vit D-IR, and Stem cell-IR groups, both ALT and AST were significantly decreased being comparable to Sham only for AST, yet still significantly higher than Sham for ALT. As a result of Vitamin D supplementation, the Combined-IR group showed a significant rise in serum level of Vit. D compared to Sham, IR, and Stem cell-IR groups with no significant difference with Vit D-IR group, also, showed significant decreases in the hepatic tissue levels of H.MDA, H.IL-2, H. Casp-3, and significant increases in H.SOD, H.IL-10, H.LC3B, H.HGF as compared to IR and Stem cell-IR groups becoming non significantly different from the Sham group. Compared to Vit. D-IR group, Combined-IR group showed a significant decrease in H.MDA, H.IL-2 and a significant increase in H.SOD, H.HGF but no significant difference regarding H.IL-10, H.LC3B. Combined-IR group showed almost complete normal hepatic morphology except for a few scattered apoptosis with the best scoring.
Moreover, fluorescence imaging of labeled BM-MSCs for the Stem cell-IR and Combined-IR groups indicated well distribution and survival of MSCs inside the liver that was more in the Combined-IR group.
In conclusion, we have demonstrated that vitamin D preconditioning is a potentially simple and cost-effective initiative to improve IR injury. Additionally, BM-MSCs represent an ideal ‘poly-pill’ therapy that could positively influence medical outcomes. The results of our study indicate that combination therapy surpassed the use of each line of treatment alone. The effects were found, through inhibition of oxidative stress and inflammatory response, augmentation of antioxidant capacity and anti-inflammatory reaction, with the enhancement of autophagy and regenerative capacity and attenuation of apoptosis. The results of this study offer new insight into the mechanisms responsible for the combined use of MSC and vitamin D-mediated protective manner.
Recommendations
The results of the present study represent experimental evidence for the beneficial effects of early administration of the combined therapy of BM-MSCs and Vitamin D3 on biochemical and histopathological derangements in hepatic ischemia-reperfusion injury induced in rats.
We focused our attention on the acute phase of hepatic IR injury and long-term effects were not investigated at this time, our intention was to better understand the mechanisms involved in their early activity. It would be interesting in future studies to evaluate harder clinical outcomes, such as longer follow-up times and survival analyses.