SB203580 Modulates p38 MAPK Signaling and Dengue Virus-Induced Liver Injury by Reducing MAPKAPK2, HSP27, and ATF2 Phosphorylation.

Dengue virus (DENV) infection causes organ injuries, and the liver is one of the most important sites of DENV infection, where viral replication generates a high viral load. The molecular mechanism of DENV-induced liver injury is still under investigation. The mitogen activated protein kinases (MAPKs), including p38 MAPK, have roles in the hepatic cell apoptosis induced by DENV. However, the in vivo role of p38 MAPK in DENV-induced liver injury is not fully understood. In this study, we investigated the role of SB203580, a p38 MAPK inhibitor, in a mouse model of DENV infection. Both the hematological parameters, leucopenia and thrombocytopenia, were improved by SB203580 treatment and liver transaminases and histopathology were also improved. We used a real-time PCR microarray to profile the expression of apoptosis-related genes. Tumor necrosis factor α, caspase 9, caspase 8, and caspase 3 proteins were significantly lower in the SB203580-treated DENV-infected mice than that in the infected control mice. Increased expressions of cytokines including TNF-α, IL-6 and IL-10, and chemokines including RANTES and IP-10 in DENV infection were reduced by SB203580 treatment. DENV infection induced the phosphorylation of p38MAPK, and its downstream signals including MAPKAPK2, HSP27 and ATF-2. SB203580 treatment did not decrease the phosphorylation of p38 MAPK, but it significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF2. Therefore, SB203580 modulates the downstream signals to p38 MAPK and reduces DENV-induced liver injury

Invalid freeze-dried platelet gel promotes wound healing

Wound healing is the curative process of tissue injury, composed of three phases: the inflammatory phase, proliferative phase, followed by the maturation cum remodeling phase. Various treatment options were previously depicted for wound healing, however a treatment that accelerates these phases would be highly valuable. Platelet aggregation at the bleeding vessels and release of various growth factors are the most promising factors that stimulates the wound healing progress. In the present study, we hypothesized that the freeze-dried platelet which were normally discarded from the blood banks due to invalidity, might be promising to accelerate the phases of wound healing. The invalid freeze-dried platelets were prepared to a gel form called invalid freeze-dried platelet gel (IF-PG), which was tested for its efficacy in a cutaneous punch wound model in rats. Mupirocin antibiotic gel was used as a bio-equivalent formulation. The wound healing phases and changes in the wound sites were determined by assessing the wound sizes, histopathological analysis, immunohistochemical staining. The re-epithelialization at the wound sites at different time intervals till the wound closure was also determined. Our results suggest the beneficial effects of IF-PG; in reducing the wound area and accelerating wound closure in the cutaneous punch wound in rats. Histopathology and immunostaining results support the improvements in the wound when treated with IF-PG, which were similar to that of mupirocin antibiotic gel. Our preliminary findings also warrant the competency of IF-PG in modulating the different phases of wound healing process. In conclusion, IF-PG might be a resourceful alternative for the wound care management, however further studies are required to validate its impact on various growth factors before proceeding to clinical studies. Keywords: IF-PG, Wound healing, Animal model, Re-epithelializatio

SB203580 treatment reduces GGT expression in DENV-infected mice.

<p>Mice were infected with 4 × 10⁵ FFU/ml of DENV and treated with 2%DMSO (v/v) or SB203580 dissolved in 2%DMSO. The control (uninfected) group was treated with 2%DMSO (v/v) alone. Treatments were given 1 h before and after DENV infection and again at 24 h after infection. On day 7 after infection, the liver tissues were collected, and the proteins were extracted for western blotting analysis using antibody directed against (A) GGT normalized to GAPDH. The results shown are representatives of three independent experiments with three mice (n = 3) from each group. A densitometry analysis using the ImageJ software is shown in (B).</p

SB203580 treatment reduces caspase 3 expression.

<p>Mice were infected with 4 × 10⁵ FFU/ml of DENV and treated with 2%DMSO (v/v) or SB203580 dissolved in 2%DMSO. The control (un-infected) group was treated with 2%DMSO (v/v) alone. Treatments were given 1 h before and after DENV infection and again at 24 h after infection. On day 7 after infection, the liver tissues were collected, and the proteins were extracted for western blotting analysis using antibodies directed against (A) pro caspase 3 and (C) cleaved caspase 3. The results shown are representative of three independent experiments with three mice (n = 3) from each group. A densitometry analysis using the ImageJ software is shown in (B) pro caspase 3 and (D) cleaved caspase 3.</p

Apoptotic gene expression profiles of DMSO-treated DENV-infected mice and SB203580 treated DENV-infected mice (both are normalized with those of DMSO-treated uninfected mice).

<p>Apoptotic gene expression profiles of DMSO-treated DENV-infected mice and SB203580 treated DENV-infected mice (both are normalized with those of DMSO-treated uninfected mice).</p

SB203580 treatment reduces caspase 8 expression.

<p>Mice were infected with 4 × 10⁵ FFU/ml of DENV and treated with 2%DMSO (v/v) or SB203580 dissolved in 2%DMSO. The control (uninfected) group was treated with 2%DMSO (v/v) alone. Treatments were given 1 h before and after DENV infection and again at 24 h after infection. On day 7 after infection, the liver tissues were collected, and the proteins were extracted for western blotting analysis using antibody directed against the pro and p18 subunit of the cleaved form of (A) caspase 8. The results shown are representative of three independent experiments with three mice (n = 3) from each group. A densitometry analysis using the ImageJ software is shown in (B).</p

SB203580 does not reduce the phosphorylation of p38 MAPK.

<p>Proteins were extracted from the liver tissue samples of 2%DMSO-treated (uninfected), 2%DMSO-treated DENV-infected, and SB203580-treated DENV-infected groups of mice. An additional cocktail of phosphatase inhibitors was added for maintaining the phosphorylated proteins and subjected to western blot analysis with specific antibodies. Results were shown for (A) phosphorylated p38 MAPK and (B) total p38 MAPK. The results shown are representative of three independent experiments with three mice (n = 3) from each group. A densitometry analysis was conducted for the individual blots, normalized to the respective GAPDH, is shown in (C) phosphorylated p38 MAPK and total p38MAPK.</p

SB203580 treatment reduces the phosphorylation of MAPKAPK2 and HSP27.

<p>Proteins were extracted from the liver tissue samples of 2%DMSO-treated (un-infected), 2%DMSO-treated DENV-infected, and SB203580-treated DENV-infected groups of mice. An additional cocktail of phosphatase inhibitors was added for maintaining the phosphorylated proteins and allowed them to standard Western blot analysis with specific antibodies. The results were shown (A) phosphorylated MAPKAPK2 and total MAPKAPK2 and (C) phosphorylated HSP27 and total HSP27, which is normalized to their respective GAPDH. The results shown are representative of three independent experiments with three mice (n = 3) from each group. A densitometry analysis was conducted with the individual blots normalized to GAPDH and is shown in (B) phosphorylated MAPKAPK2 and total MAPKAPK2 and (D) phosphorylated HSP27 and total HSP27.</p

SB203580 treatment reduces liver enzymes in DENV-infected mice.

<p>Mice were infected with DENV and treated with 2%DMSO or SB203580. The uninfected control group was also treated with 2%DMSO. Seven days after infection, blood was collected and sera prepared to estimate liver transaminases. (A) ALT, and (B) AST. The pooled results of two independent experiments are presented as means ± SEM (12 mice per group in two independent experiments). The asterisks indicate statistically significant differences between the groups (<i>p</i> < 0.05).</p

SB203580 modulates the cytokine and chemokine gene expressions in DENV infection

<p>Mice were infected with 4 × 10⁵ FFU/ml of DENV and treated with 2%DMSO (v/v) or SB203580 dissolved in 2%DMSO. The control (uninfected) group was treated with 2%DMSO (v/v) alone. Treatments were given 1 h before and after DENV infection and again at 24 h after infection. On day 7 after infection, the liver tissues were collected, and RNA was extracted, cDNA were prepared and which undergone Real-time RT PCR analysis with individual primer set. GAPDH is used as the house keeping gene. The mRNA expression of (A) TNF-α (B) IL-6 (C) IL-10 (D) CCL-5 (E) CXCL-10 are shown. Results were represented in the graph by three independent experiments for at least 3 independent mice from each group. Statistical analysis is conducted by One Way ANOVA using GraphPad Prism Software Version 5. The asterisks indicate statistically significant differences between groups (<i>p</i>< 0.05).</p