Centella asiatica (Gotu kola) ethanol extract up-regulates hippocampal brain-derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) signaling in chronic electrical stress model in rats.

OBJECTIVES: Impairment of hippocampus function as a center for memory processing occurs due to stress. Centella asiatica L. (Gotu kola) is known to improve memory, intelligence, and neural protection although the precise mechanism is not well understood. This study aimed to investigate the effects of ethanol extracts of C. asiatica toward MAPK expression as down-stream signaling of brain-derived neurotrophic factor (BDNF). MATERIALS AND METHODS: We performed a chronic electrical stress model on 20 male Sprague Dawley rats (2 months-old, 180-200 g). Rats were divided into four groups: normal control group (Control) which received distilled water, and three treatment groups receiving oral Gotu kola ethanol extracts in oral doses of 150 mg/kg BW (CeA150), 300 mg/kg BW (CeA300), and 600 mg/kg BW (CeA600) over four weeks. Memory acquisition was assessed with Morris water maze. Hippocampus was harvested, then extracted for protein and RNA analysis. MAPK proteins (p38, ERK1/2, JNK) were measured using Western blot, meanwhile, BDNF and TrkB receptor were analyzed with real-time PCR (RT-PCR). RESULTS: CeA600 group revealed improvement of memory performance as shown by reduction in time and distance parameters compared to control during escape latency test. This finding associated with significant elevation of hippocampal BDNF protein and mRNA level with up-regulation of TrkB mRNA expression in CeA600 group compared to control. Western-blot analysis showed significant up-regulation of ERK1/2 protein level in CeA600 group (P<0.05) compare to control. CONCLUSION: BDNF signaling through TrkB and ERK1/2 pathway contributes significantly to amelioration of memory performance after C. asiatica treatment in electrical stress model.

Hyperuricemia Induces Wnt5a/Ror2 Gene Expression, Epithelial-Mesenchymal Transition, and Kidney Tubular Injury in Mice.

BACKGROUND: Hyperuricemia contributes to kidney injury, characterized by tubular injury with epithelial-mesenchymal transition (EMT). Wnt5a/Ror2 signaling drives EMT in many kidney pathologies. This study sought to evaluate the involvement of Wnt5a/Ror2 in hyperuricemia-induced EMT in kidney tubular injury. METHODS: A hyperuricemia model was performed in male Swiss background mice (3 months old, 30-40 g) with daily intraperitoneal injections of 125 mg/kg body weight (BW) of uric acid. The mice were terminated on day 7 (UA7, n=5) and on day 14 (UA14, n=5). Allopurinol groups (UAl7 and UAl14, each n=5) were added with oral 50 mg/kg BW of allopurinol treatment. The serum uric acid level was quantified, and tubular injury was assessed based on PAS staining. Reverse transcriptase-PCR was done to quantify Wnt5a, Ror2, E-cadherin, and vimentin expressions. IHC staining was done for E-cadherin and collagen I. We used the Shapiro-Wilk for normality testing and one-way ANOVA for variance analysis with a P<0.05 as significance level using SPSS 22 software. RESULTS: The hyperuricemia groups had a higher uric acid level, which was associated with a higher tubular injury score. Meanwhile, the allopurinol groups had a significantly lower uric acid level and tubular injury than the uric acid groups. Reverse transcriptase-PCR revealed downregulation of the E-cadherin expression. While vimentin and collagen I expression are upregulated, which was associated with a higher Wnt5a expression. However, the allopurinol groups had reverse results. Immunostaining revealed a reduction in E-cadherin staining in the epithelial cells and collagen I positive staining in the epithelial cells and the interstitial areas. CONCLUSION: Hyperuricemia induced tubular injury, which might have been mediated by EMT through the activation of Wnt5a.

Uric acid causes kidney injury through inducing fibroblast expansion, Endothelin-1 expression, and inflammation.

BACKGROUND: Uric acid (UA) plays important roles in inducing renal inflammation, intra-renal vasoconstriction and renal damage. Endothelin-1 (ET-1) is a well-known profibrotic factor in the kidney and is associated with fibroblast expansion. We examined the role of hyperuricemia conditions in causing elevation of ET-1 expression and kidney injury. METHODS: Hyperuricemia was induced in mice using daily intraperitoneal injection of uric acid 125 mg/Kg body weight. An NaCl injection was used in control mice. Mice were euthanized on days-7 (UA7) and 14 (UA14). We also added allopurinol groups (UAL7 and UAL14) with supplementation of allopurinol 50 mg/Kg body weight orally. Uric acid and creatinine serum were measured from blood serum. Periodic Acid Schiff (PAS) and Sirius Red staining were done for glomerulosclerosis, tubular injury and fibrosis quantification. mRNA expression examination was performed for nephrin, podocin, preproEndothelin-1 (ppET-1), MCP-1 and ICAM-1. PDGFRß immunostaining was done for quantification of fibroblast, while α-SMA immunostaining was done for localizing myofibroblast. Western blot analysis was conducted to quantify TGF-ß1, α-SMA and Endothelin A Receptor (ETAR) protein expression. RESULTS: Uric acid and creatinine levels were elevated after 7 and 14 days and followed by significant increase of glomerulosclerosis and tubular injury score in the uric acid group (p < 0.05 vs. control). Both UA7 and UA14 groups had higher fibrosis, tubular injury and glomerulosclerosis with significant increase of fibroblast cell number compared with control. RT-PCR revealed down-regulation of nephrin and podocin expression (p < 0.05 vs. control), and up-regulation of MCP-1, ET-1 and ICAM-1 expression (p < 0.05 vs. control). Western blot revealed higher expression of TGF-ß1 and α-SMA protein expression. Determination of allopurinol attenuated kidney injury was based on reduction of fibroblast cell number, inflammation mediators and ppET-1 expression with reduction of TGF-ß1 and α-SMA protein expression. CONCLUSIONS: UA induced glomerulosclerosis, tubular injury and renal fibrosis with reduction of podocyte function and inflammatory mediator elevation. ET-1 and fibroblast expansion might modulate hyperuricemia induced renal fibrosis.

Vitamin D Attenuates Kidney Fibrosis via Reducing Fibroblast Expansion, Inflammation, and Epithelial Cell Apoptosis.

Kidney fibrosis is the common final pathway of chronic kidney diseases (CKD). It is characterized by myofibroblast formation, inflammation, and epithelial architecture damage. Vitamin D is known as a renoprotective agent, although the precise mechanism is not well understood. This study aimed to elucidate the effect of vitamin D in fibroblast expansion, inflammation, and apoptosis in kidney fibrosis. We performed unilateral ureteral obstruction (UUO) in male Swiss-Webster background mice (3 months, 30-40 grams) to induce kidney fibrosis. The mice (n=25) were divided into five groups: UUO, 3 groups treated with different oral vitamin D doses (0.125 µg/kg (UUO+VD1), 0.25 µg/kg (UUO+VD2), and 0.5 µg/kg (UUO+VD3), and a Sham operation (SO) group with ethanol 0.2% supplementation. We sacrificed the mice on day14 after the operation and harvested the kidney. We made paraffin sections for histological analysis. Tubular injury and fibrosis were quantified based on periodic acid-Schiff (PAS) and Sirius Red (SR) staining. Immunostaining was done for examination of myofibroblasts (αSMA), fibroblasts (PDGFRß), TLR4, and apoptosis (TUNEL). We did RNA extraction and cDNA for Reverse transcriptase PCR (RT-PCR) experiment for measuring MCP-1, ICAM-1, TLR4, and collagen 1 expression. TGFß1 level was quantified using ELISA. We observed a significantly lower levels of fibrosis (p<0.001), tubular injury scores (p<0.001), and myofibroblast areas (p<0.001) in the groups treated with vitamin D compared with the UUO group. The TGFß1 levels and the fibroblast quantifications were also significantly lower in the former group. However, we did not find any significant difference among the various vitamin D-treated groups. Concerning the dose-independent effect, we only compared the UUO+VD-1 group with SO group and found by TUNEL assay that UUO+VD-1 had a significantly lower epithelial cell apoptosis. RT-PCR analysis showed lower expression of collagen1, as well as inflammation-mediator expression (MCP-1, ICAM-1, TLR4) in the UUO+VD-1 group compared with the SO group. Vitamin D reduces kidney fibrosis through inhibition of fibroblast activation, and ameliorates epithelial cell architecture.