Effect of Hemin on Brain Alterations and Neuroglobin Expression in Water Immersion Restraint Stressed Rats

In the brain, the heme oxygenase (HO) system has been reported to be very active and its modulation seems to play a crucial role in the pathophysiology of neurodegenerative disorders. Hemin as HO-1 inducer has been shown to attenuate neuronal injury so the goal of this study was to assess the effect of hemin therapy on the acute stress and how it would modulate neurological outcome. Thirty male albino rats were divided into three groups: control group and stressed group with six-hour water immersion restraint stress (WIRS) and stressed group, treated with hemin, in which each rat received a single intraperitoneal injection of hemin at a dose level of 50 mg/kg body weight at 12 hours before exposure to WIRS. Stress hormones, oxidative stress markers, malondialdehyde (MDA), and total antioxidant capacity (TAC) were measured and expressions of neuroglobin and S100B mRNA in brain tissue were assayed. Our results revealed that hemin significantly affects brain alterations induced by acute stress and this may be through increased expression of neuroglobin and through antioxidant effect. Hemin decreased blood-brain barrier damage as it significantly decreased the expression of S100B. These results suggest that hemin may be an effective therapy for being neuroprotective against acute stress

Investigating the Relationship between Insulin-like Growth Factor-1 (IGF-1) in diabetic mother’s breast milk and the blood serum of their babies

Introduction: Since research investigating IGF-1 levels in breast milk are few, the goal of this study was to analyze the IGF-1 levels in the breast milk of diabetic mothers as well as in the serum of their newborn babies and to identify what relationship exists between blood serum and IGF-1 milk levels through patient measurement of mothers and their babies. Methods: This case control study was undertaken under the auspices of the Clinic of Neonatology at Al Minia University Pediatric Hospital over May 2012 through May 2013. With a total of 30 diabetic mothers and their babies forming the experimental group and the control group consisting of 15 non-diabetic mothers and their babies. A detailed medical history, anthropometric assessments, as well as the measurement of the baby’s serum IGF-1 and their mother’s breast milk IGF-1 levels were taken from all participants using ELSIA. The resulting data were analyzed via Statistical Package for the Social Sciences (SPSS) version 16 and measurements of descriptive statistics, t-test, Chi-square test, as well as the Pearson Correlation Coefficient. Results: The Infants born to Diabetic Mothers (IDMs) demonstrated significantly greater anthropometric measurement. Both the serum levels and the milk IGF-1 levels as well as all of the physical measurements taken were found to have a positive correlation between the level of IGF-1 in mother’s milk and all of the anthropometric measurements studied with the exception of delivered baby’s length. Conclusion: Higher levels of IGF-1 are present in the milk of diabetic mothers and the blood serum of their babies and this characteristic could be used as a prenatal biomarker for macrosomia

Mutant MMP-9 and HGF Gene Transfer Enhance Resolution of CCl₄-Induced Liver Fibrosis in Rats: Role of ASH1 and EZH2 Methyltransferases Repression

<div><p>Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl₄ induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl₄ for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. Conclusion: Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and EZH2 in resolution of liver fibrosis.</p></div

Effect of gene therapy on molecular markers of liver fibrosis.

<p>(A, B) Col1α1 mRNA expression was significantly elevated (*, p<0.001) in saline-treated livers compared with healthy livers (n = 10), and was significantly reduced with HGF (n = 14, #, p<0.05), mMMP-9 (n = 10, *, p<0.001) and HGF/mMMP-9 (n = 8, p<0.001) treatments compared with saline-treated fibrotic livers (n = 13). (C, D) α-SMA mRNA expression was significantly reduced with HGF (n = 14, p<0.001), mMMP-9 (n = 10, p<0.001) and HGF/mMMP-9 (n = 9, p<0.001) treatments compared with saline-treated fibrotic livers and was significantly elevated (p<0.001) in saline-treated fibrotic livers compared with healthy livers (n = 10). (E, F) TGF-β1 mRNA expression was significantly reduced with mMMP-9 (n = 10, p<0.01) and HGF/mMMP-9 (n = 9, p<0.01) treatment but not with HGF (n = 13, p = 0.87) compared with saline-treated fibrotic livers (n = 13). (G) TIMP-1 protein concentration was elevated (p<0.01) in saline-treated fibrotic livers (n = 9) and was further elevated in HGF-treated livers (n = 13) compared with healthy liver (n = 9). TIMP-1 concentration in fibrotic livers was significantly (p<0.001) decreased with mMMP-9 (n = 10) but not with HGF (p = 0.09) or with HGF/mMMP-9 (n = 9, p = 0.69) treatment. (H) Hydroxyproline concentration in saline-treated fibrotic livers (n = 10) was significantly elevated (p<0.001) compared with healthy livers (n = 10) and was significantly (p<0.001) decreased only with mMMP-9 treatment (n = 10) but not with HGF (n = 11, p = 0.86) or with HGF/mMMP-9 (n = 7, p = 0.98) treatments. (I) ALT serum concentration in saline-treated fibrotic rats (n = 14) was significantly elevated (p<0.001) compared with healthy rats (n = 10) and was significantly decreased with mMMP-9 (n = 10, p<0.001) and combined HGF/mMMP-9 (n = 9, p<0.01) but not with HGF (n = 14, p = 0.15) treatment.</p

Analysis of ASH1 and EZH2 methyltransferases protein expression with Slot Blot.

<p>ASH1 (A, B) and EZH2 (C, D) protein expression were undetectable in the healthy livers. Both ASH1 and EZH2 protein expression markedly increased (p<0.01) following CCl₄ treatment for eight weeks. Treatment with HGF, mMMP-9 or combined HGF/mMMP-9 resulted in significant (p<0.01) reduction of both ASH1 and EZH2 protein expression in fibrotic livers. There was no significant difference in the ASH1 or EZH2 protein expression between the HGF-, mMMP-9-, or HGF/mMMP-9-treated fibrotic livers. (n = 6 per group).</p

Immunohistochemical staining for α-SMA and PCNA in the liver.

<p>Bar graphs represent α-SMA and PCNA labeling indices (percent of positively stained brown cytoplasm or brown nuclei, respectively in 10 successive fields). Both α-SMA and PCNA indices were significantly elevated (*, p<0.001) in saline-treated fibrotic (n = 14 both) compared with healthy livers (n = 10 both) and both were significantly reduced (*, p<0.001) in mMMP-9-treated (n = 10 both) and in HGF/mMMP-9-treated (n = 9 both, *, p<0.001 and #, p<0.01, respectively) versus saline-treated fibrotic livers. Both α-SMA and PCNA labeling indices were not significantly different in HGF-treated fibrotic (n = 14, p = 0.99 and p = 0.06, respectively) from that in saline-treated fibrotic livers. (Original magnification ×400).</p

Histopathological findings in rat liver sections stained with H&E and Sirius-red.

<p>Fibrosis score was significantly elevated (*, p<0.001) in saline-treated fibrotic (n = 14) compared with healthy livers (n = 10). Fibrosis scores of HGF-treated livers (n = 14) were not statistical different from those of saline-treated livers (p = 0.71). However, fibrosis score was significantly reduced (**, p<0.01) in mMMP-9-treated (n = 9) and (#, p<0.05) in HGF/mMMP-9-treated (n = 9) compared to saline-treated fibrotic livers. (Original magnification ×200). Sirius red stained collagen area percent was significantly elevated (p<0.001) in saline-treated fibrotic livers (n = 14) compared with healthy livers (n = 10). Collagen area percent was significantly reduced in mMMP-9-treated (n = 9, p<0.001)) and in HGF/mMMP-9-treated (n = 9, p<0.01) but not in HGF-treated (n = 13, p = 0.19) compared to saline-treated fibrotic livers. (Original magnification, ×100).</p

Analysis of HGF and MMP-9 gene expression.

<p>(A, B) RT-PCR gel of HGF mRNA expression. HGF mRNA expression was significantly (*, p<0.001) elevated in the saline-treated fibrotic (n = 14) compared to saline-treated healthy livers (n = 10) and further elevated (#, p<0.01) in the HGF-treated fibrotic (n = 13) compared with the saline-treated fibrotic and mMM-9-treated fibrotic (n = 10) livers (*, p<0.01). (C) Analysis of HGF protein concentration in the liver tissues. HGF protein concentration was significantly (*, p<0.001) elevated in the liver of the saline-treated fibrotic livers (n = 11) compared to saline-treated healthy livers (n = 10) and more elevated in the HGF-treated fibrotic (n = 14) compared to the saline-treated fibrotic (#, p<0.05) and mMMP-9-treated fibrotic (n = 10) livers (**, p<0.01). (D) Analysis of MMP-9 protein concentration in the liver tissues. MMP-9 protein concentration was significantly elevated in the mMMP-9-treated (n = 10) compared with healthy (n = 9), saline-treated fibrotic (n = 13) livers, HGF-treated (n = 12) livers (*, p<0.01) and HGF/mMMP-9-treated (n = 6) livers (#, p<0.05).</p