Pirfenidone and Vitamin D Ameliorate Cardiac Fibrosis Induced by Doxorubicin in Ehrlich Ascites Carcinoma Bearing Mice: Modulation of Monocyte Chemoattractant Protein-1 and Jun N-terminal Kinase-1 Pathways

Treatment of breast cancer with doxorubicin causes numerous side effects, of which cardiac fibrosis is considered the main one. This study was designed to investigate the underlying molecular mechanisms for the potential anti-fibrotic effect of pirfenidone and vitamin D against doxorubicin-induced cardiac fibrosis. Seventy mice carrying solid Ehrlich’s ascites carcinoma (EAC) discs on the ventral side were treated with orally administered pirfenidone (500 mg/kg) and intraperitoneal injection of vitamin D (0.5 µg/kg) either individually or in combination with a doxorubicin (15 mg/kg; i.p.) single dose. All treatments commenced one week post-tumor inoculation and continued for 14 days. Compared to control EAC mice, the doxorubicin group showed a significant increase in heart and left ventricle weights, troponin T, and creatinine kinase serum levels. Furthermore, the doxorubicin group depicts a high expression of monocyte chemoattractant protein (MCP-1), nuclear factor-kappa B (NF-κB), transforming growth factor-beta 1 (TGF-β1), smad3, Jun N-terminal Kinase-1 (JNK1), and alpha-smooth muscle actin (α-SMA). Treatment with pirfenidone or vitamin D significantly decreased all of these parameters. Furthermore, the expression of smad7 was downregulated by doxorubicin and improved by pirfenidone or vitamin D. Furthermore, all treated groups showed a marked decrease in tumor weight and volume. Current data demonstrate that pirfenidone and vitamin D represent an attractive approach to ameliorate the cardiac fibrosis produced by doxorubicin through inhibiting both JNK1 signaling and MCP-1 inflammatory pathways, thus preserving heart function. Further, this combination demonstrated an anti-tumor effect to combat breast cancer

Pioglitazone Ameliorates Hippocampal Neurodegeneration, Disturbances in Glucose Metabolism and AKT/mTOR Signaling Pathways in Pentyelenetetrazole-Kindled Mice

Disturbance of glucose metabolism, nerve growth factor (NGF) and m-TOR signaling have been associated with the pathophysiology of epilepsy. Pioglitazone (PGZ) is an anti-diabetic drug that shows a protective effect in neurodegenerative diseases including epilepsy; however, its exact mechanism is not fully elucidated. The present study aimed to investigate the potential neuroprotective effect of PGZ in pentylenetetrazole (PTZ) kindled seizure in mice. Swiss male albino mice were randomly distributed into four groups, each having six mice. Group 1 was considered the control. Epilepsy was induced by PTZ (35 mg/kg i.p.) thrice a week for a total of 15 injections in all other groups. Group 2 was considered the untreated PTZ group while Group 3 and Group 4 were treated by PGZ prior to PTZ injection at two dose levels (5 and 10 mg/kg p.o., respectively). Seizure activity was evaluated after each PTZ injection according to the Fischer and Kittner scoring system. At the end of the experiment, animals were sacrificed under deep anesthesia and the hippocampus was isolated for analysis of glucose transporters by RT-PCR, nerve growth factor (NGF) by ELISA and mTOR by western blotting, in addition to histopathological investigation. The PTZ-treated group showed a significant rise in seizure score, NGF and m-TOR hyperactivation, along with histological abnormalities compared to the control group. Treatment with PGZ demonstrated a significant decrease in NGF, seizure score, m-TOR, GLUT-1 and GLUT-3 in comparison to the PTZ group. In addition, improvement of histological features was observed in both PGZ treated groups. These findings suggest that PGZ provides its neuroprotective effect through modulating m-TOR signaling, glucose metabolism and NGF levels

Diabetes and overexpression of proNGF cause retinal neurodegeneration via activation of RhoA pathway.

Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotocin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75(NTR), cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75(NTR) and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75(NTR) expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways

Inhibiting Rho kinase blocked proNGF and diabetes-induced retinal neurodegeneration.

<p><b>A,B.</b> Representative images and statistical analysis of TUNEL-HRP-positive cells counted in each retina flat-mount showing ∼9-fold increased number of cell death in retinas from 5 weeks diabetic rats as compared with the controls (n = 4–5). <b>C.</b> Statistical analysis of total number of TUNEL-HRP-positive cells counted in each retina showing ∼6-fold increase of cell death in retinas that overexpress proNGF as compared with the GFP controls (n = 4–5). Treatment with the selective Rho kinase inhibitor Y27632 blocked these effects in diabetic and proNGF overexpression and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control; D, diabetic; Y, Y27632.</p

Diagram depicting the proposed role of proNGF/p75^NTR in diabetic retinopathy.

<p>Retinal neurodegeneration is thought to be induced via direct activation of RhoA kinase in RGC and paracrine inflammatory action in response to increases in proNGF.</p

Inhibiting Rho kinase blocked diabetes- and proNGF-induced apoptotic markers expression.

<p><b>A,C.</b> WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in rats electroporated with proNGF as compared with the controls (n = 4–5). <b>B,D.</b> WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 cells treated with proNGF as compared with the controls (n = 4). <b>E.</b> WB analysis showing 2.1- and 1.6-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 treated with proNGF as compared with the controls. Treatment of rats or RGC-5 with Y27632 blocked all these effects in rats and media treated with proNGF and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control.</p

ProNGF selectively activates RhoA kinase activation in vivo and in RGC cultures.

<p><b>A.</b> Pull-down assay of rat retinal lysate showed 2.3-fold increase in the expression of active Rho in diabetic rats as compared with the controls (n = 4–5). <b>B.</b> Pull-down assay of rat retinal lysate showed 1.7-fold increase in active RhoA expression in rats electroporated with proNGF as compared with those electroporated with GFP (n = 5). <b>C.</b> Pull-down assay of RGC-5 lysate showed 1.6-fold increase in RhoA expression in RGC-5 cells treated with proNGF as compared with the controls (n = 4). Treatment of rats or RGC-5 with Y27632 blocked RhoA activation proNGF-treated samples but not the control groups. <b>D.</b> Pull-down assay of RGC-5 showing that treatment of RGC-5 cells with TNF-α did not increase RhoA activation as compared with the control group (n = 4). <b>E.</b> Statistical analysis showing overexpression of proNGF in healthy retina induced 3-fold increase in TNF-α mRNA expression as compared with the control group. <b>F.</b> Statistical analysis of G-LISA showing 1.7-fold increase of RhoA in primary RGC cultures treated with proNGF as compared with the control. TNF-α caused modest increase in RhoA activation (20%) as compared with the controls. These effects were reduced by treatment with Y27632. * = significant difference as compared with the control group at p<0.05.</p

Diabetes and overexpression of proNGF induced expression of p75^NTR in vivo and in vitro.

<p><b>A.</b> WB analysis showing 1.9-fold increase in the expression of p75^NTR in diabetic rats as compared with the controls (n = 4–6). <b>B.</b> WB analysis of rat retinal lysate showed significant increase in p75^NTR expression in rats electroporated with proNGF as compared with those electroporated with GFP (n = 4). <b>C.</b> Representative images of rat retina sections showing prominent immunolocalization of p75^NTR in GCL and INL in proNGF overexpressing retinas as compared with GFP-controls (400× magnification). <b>D.</b> Representative images of rat retina sections showing colocalization between p75^NTR in the ganglion cell layer (green) and the specific neuronal marker Thy-1 (red) in the upper pannel or with the specific RGC marker Brn-3a (red)in the lower pannel (400× magnification). <b>E.</b> Western blot analysis shwoing 1.6-fold increase in the expression of p75^NTR in RGC-5 cells treated with proNGF as compared with the controls (n = 4). <b>F.</b> Real-time PCR analysis showing that proNGF induced p75^NTR mRNA expression in freshly isolated primary RGC as compared with the control group. Samples of primary RGC cultures were pooled from 4-different cultures. Treatment with Y27632 significantly reduced p75^NTR expression <i>in vivo</i> and <i>in vitro</i>. G. Representative images showing colocalization (yellow arrow heads) of RGC that expressed p75^NTR (red) and the specific RGC marker Brn3a (green) in isolated mixed neuronal cultures from adult mice. * = significant difference as compared with the rest of the groups at p<0.05. C, control; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. C, control.</p