Chih-Hsien Chiu

Curcumin protects against thioacetamide-induced hepatic fibrosis by attenuating the inflammatory response and inducing apoptosis of damaged hepatocytes

Inflammation and hepatic stellate cell (HSC) activation are the most crucial steps in the formation of hepatic fibrosis. Hepatocytes damaged by viral or bacterial infection, alcohol or toxic chemicals initiate an inflammatory response that activates collagen production by HSCs. Recent studies indicate curcumin has liver-protective effects due to its anti-inflammatory, antioxidant and anticancer activities; however, the mechanisms are not well understood. In this study, we show that curcumin protected against hepatic fibrosis in BALB/c mice in vivo by inhibiting HSC activation, inflammatory responses and inducing apoptosis of damaged hepatocytes. Using the thioacetamide (TAA)-induced hepatic fibrosis animal model, we found that curcumin treatment up-regulated P53 protein expression and Bax messenger RNA (mRNA) expression and down-regulated Bcl-2 mRNA expression. Together, these responses increased hepatocyte sensitivity to TAA-induced cytotoxicity and forced the damaged cells to undergo apoptosis. Enhancing the tendency of damaged hepatocytes to undergo apoptosis may be the protective mechanism whereby curcumin suppresses inflammatory responses and hepatic fibrogenesis. These results provide a novel insight into the cause of hepatic fibrosis and the cytoprotective effects curcumin has on hepatic fibrosis suppression.

Hepatoprotection of silymarin against thioacetamide-induced chronic liver fibrosis

BACKGROUND Liver fibrosis is chronic liver damage usually caused by alcohol, viruses or other toxins and is characterised by an excessive accumulation of extracellular matrix proteins such as collagen. The aim of this study was to establish an animal model of chronic liver damage and investigate molecular mechanisms of silymarin hepatoprotective effects. RESULTS Thioacetamide (TAA; 100 mg kg(-1) intraperitoneal (i.p.) injection three times weekly) effectively induced chronic liver fibrosis in male ICR mice. Then 24 ICR mice were randomly divided into four groups: (1) saline (i.p.) + water (gavage); (2) saline (i.p.) + 150 mg kg(-1) silymarin (gavage); (3) 100 mg kg(-1) TAA (i.p.) + water (gavage); (4) 100 mg kg(-1) TAA (i.p.) + 150 mg kg(-1) silymarin (gavage). Eight weeks of TAA treatment resulted in lower body weight, serum cholesterol and triglycerides as well as increased liver size, ALT, AST and LDH values (P < 0.05). These TAA-induced effects were attenuated by silymarin (P < 0.05); therefore silymarin also ameliorated TAA-induced liver lesions. Effects of silymarin on TAA-induced chronic liver damage may be attributed to down-regulation of hepatic MMP-2, MMP-13, TIMP-1, TIMP-2, AP-1, KLF6, TGF-β1, α-SMA and COL-α1. CONCLUSION A mouse model of chronic liver fibrosis was successfully established by injecting 100 mg kg(-1) TAA three times weekly in male ICR mice. Meanwhile, silymarin showed hepatoprotection against TAA-induced damage.

Kisspeptin modulates fertilization capacity of mouse spermatozoa

Kisspeptin acts as an upstream regulator of the hypothalamus-pituitary-gonad axis, which is one of the main regulatory systems for mammalian reproduction. Kiss1 and its receptor Kiss1r (also known as G protein-coupled receptor 54 (Gpr54)) are expressed in various organs, but their functions are not well understood. The purpose of this study was to investigate the expression profiles and functions of kisspeptin and KISS1R in the reproductive tissues of imprinting control region mice. To identify the expression pattern and location of kisspeptin and KISS1R in gonads, testes and ovarian tissues were examined by immunohistochemical or immunofluorescent staining. Kisspeptin and KISS1R were expressed primarily in Leydig cells and seminiferous tubules respectively. KISS1R was specifically localized in the acrosomal region of spermatids and mature spermatozoa. Kisspeptin, but not KISS1R, was expressed in the cumulus-oocyte complex and oviductal epithelium of ovarian and oviductal tissues. The sperm intracellular calcium concentrations significantly increased in response to treatment with kisspeptin 10 in Fluo-4-loaded sperm. The IVF rates decreased after treatment of sperm with the kisspeptin antagonist peptide 234. These results suggest that kisspeptin and KISS1R might be involved in the fertilization process in the female reproductive tract. In summary, this study indicates that kisspeptin and KISS1R are expressed in female and male gametes, respectively, and in mouse reproductive tissues. These data strongly suggest that the kisspeptin system could regulate mammalian fertilization and reproduction.

Hypoxia regulates cell proliferation and steroidogenesis through protein kinase A signaling in bovine corpus luteum.

Hypoxia is an important physiological process which ensures corpus luteum (CL) formation and development, thus playing an important role in steroidogenesis. Recent studies have shown that CL develops in an analogous to tumorigenesis by accumulation of hypoxia-inducible factor-1 alpha subunit (HIF1A) in response to hypoxia. To investigate the relationship among hypoxia, steroidogenesis, and cell proliferation during CL lifespan, histological and steroidogenic analyses of CL were performed at various CL stages in non-pregnant Holstein. Also, the hypoxia-mediated steroidogenesis and cell proliferation were studied in vitro with both primary luteal and luteinized granulosa cells. Our results showed that progesterone (P(4)) concentration increased with the upregulation of steroidogenic protein including steroidogenic acute regulatory protein (STAR) and CYP11A1 (P450scc) in the middle luteal stage. On the other hand, the cell proliferation- or hypoxia-associated proteins were upregulated in the early stage, including the proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor A (VEGFA), HIF1A, and aryl hydrocarbon receptor nuclear translocator (ARNT). In primary culture, phospho-protein kinase A (p-PKA) was downregulated, as were P(4) secretion and steroidogenic proteins both under oxygen-conditioned hypoxia in luteal cells and cobalt chloride-induced hypoxia in luteinized granulosa cells. However, under the treatment of hypoxia, PCNA, which was downregulated in luteal cells, was upregulated together with HIF1A and VEGFA in luteinized granulosa cells. Taken together, present study suggested that hypoxia downregulated steroidogenesis through PKA signaling and that the hypoxia-regulated cell proliferation could be activated during CL formation.

Effects of Antrodia camphorata on alcohol clearance and antifibrosis in livers of rats continuously fed alcohol.

Alcoholic fatty liver disease (AFLD) is the result of an excessive or chronic consumption of alcohol. Nine male Wistar rats per group were randomly assigned to one of the following drinking treatments: a 20% (w/w) alcohol solution (ALC); a 20% (w/w) alcohol solution cotreated with 0.25 g silymarin/kg BW/day; or a 20% (w/w) alcohol solution cotreated with 0.025 g Niuchangchih ( Antrodia camphorata )/kg BW/day for 4 weeks. Rats with cotreatments of silymarin or Niuchangchih had smaller (p < 0.05) relative liver size, less (p < 0.05) liver lipid accumulation, and lower (p < 0.05) liver damage indices [aspartate aminotransferase (AST) and alkaline phosphatase (ALP) values]. In the regulation of alcohol metabolism, the lower serum alcohol level was observed only in alcohol-fed rats supplemented with Niuchangchih. Meanwhile, cotreatment of silymarin or Niuchangchih increased (p < 0.05) CAT and ALDH activities but did not (p > 0.05) affect ADH and CYP2E1 expressions, which accelerate alcohol metabolism in the body. Additionally, neither silymarin nor Niuchangchih (p > 0.05) influenced serum/hepatic MMP-2 activities and NF-κB, AP1, and α-SMA gene expressions, but serum/hepatic MMP-9 activities and TNF-α, KLF-6, and TGF-β1 gene expressions of alcohol-fed rats were down-regulated (p < 0.05) by silymarin or Niuchangchih, which also could explain the lower liver damage observed in rats chronically fed alcohol.

A time-course study of gene responses of chicken granulosa cells to Salmonella Enteritidis infection

Consumption of eggs contaminated with Salmonella Enteritidis (SE) has been recognized as one of the important causes of human foodborne salmonellosis. Chicken granulosa cells (cGCs) comprise the last tissue layer surrounding the yolk in preovulatory follicles and are a preferred site for SE invasion. To understand the cGC response to SE infection, we conducted an in vitro time-course study to identify cGC transcriptional changes using chicken whole genome microarrays. The expression of 135 (4h postinfection) and 120 cGC genes (48 h postinfection) were altered (P<.01) compared to uninfected cells. Many of the altered genes were related to immune response, physiological processes, signal transduction, and transcription. Furthermore, we also found that the Jak-STAT pathway, which is essential in the regulation of cellular cytokines and growth factors, was highly active in this study. Among the genes identified by microarray, the mRNA levels of TLR15, IL-6, CXCLi1, CXCLi2, and K203 were shown to be upregulated by real-time RT-PCR (qRT-PCR). In contrast, the mRNA levels of RASD1 and HB-EGF decreased according to both microarray and qRT-PCR analyses. These results suggest that during the SE infection, cGCs recruit cells of the innate immune responses; the infection may also induce suppression of cGC cell proliferation, which alters follicular development and ovulation.

Astaxanthin Protects Steroidogenesis from Hydrogen Peroxide-Induced Oxidative Stress in Mouse Leydig Cells

Androgens, especially testosterone produced in Leydig cells, play an essential role in development of the male reproductive phenotype and fertility. However, testicular oxidative stress may cause a decline in testosterone production. Many antioxidants have been used as reactive oxygen species (ROS) scavengers to eliminate oxidative stress to protect steroidogenesis. Astaxanthin (AST), a natural extract from algae and plants ubiquitous in the marine environment, has been shown to have antioxidant activity in many previous studies. In this study, we treated primary mouse Leydig cells or MA-10 cells with hydrogen peroxide (H2O2) to cause oxidative stress. Testosterone and progesterone production was suppressed and the expression of the mature (30 kDa) form of StAR protein was down-regulated in MA-10 cells by H2O2 and cAMP co-treatment. However, progesterone production and expression of mature StAR protein were restored in MA-10 cells by a one-hour pretreatment with AST. AST also reduced ROS levels in cells so that they were lower than the levels in untreated controls. These results provide additional evidence of the potential health benefits of AST as a potential food additive to ease oxidative stress.

Cardiovascular protection of deep-seawater drinking water in high-fat/cholesterol fed hamsters

Cardiovascular protection of deep-seawater (DSW) drinking water was assessed using high-fat/cholesterol-fed hamsters in this study. All hamsters were fed a high-fat/cholesterol diet (12% fat/0.2% cholesterol), and drinking solutions were normal distiled water (NDW, hardness: 2.48ppm), DSW300 (hardness: 324.5ppm), DSW900 (hardness: 858.5ppm), and DSW1500 (hardness: 1569.0ppm), respectively. After a 6-week feeding period, body weight, heart rates, and blood pressures of hamsters were not influenced by DSW drinking waters. Serum total cholesterol (TC), triacylglycerol (TAG), atherogenic index, and malondialdehyde (MDA) levels were decreased (p<0.05) in the DSW-drinking-water groups, as compared to those in the NDW group. Additionally, increased (p<0.05) serum Trolox equivalent antioxidant capacity (TEAC), and faecal TC, TAG, and bile acid outputs were measured in the DSW-drinking-water groups. Hepatic low-density-lipoprotein receptor (LDL receptor) and cholesterol-7α-hydroxylase (CYP7A1) gene expressions were upregulated (p<0.05) by DSW drinking waters. These results demonstrate that DSW drinking water benefits the attenuation of high-fat/cholesterol-diet-induced cardiovascular disorders in hamsters.

Generation and Utilization of P450 Cholesterol Side‐Chain Cleavage Enzyme and 3β‐Hydroxysteroid Dehydrogenase Antibodies for Universal Detection

Abstract The biosynthesis of steroids from steroidogenic cells are catalyzed by the two major enzymes, P450 side‐chain cleavage enzyme (P450scc) and 3β‐hydroxysteroid dehydrogenase (3β‐HSD). This article describes the construction of two novel polyclonal antibodies against conserved recombinant protein and the validation of these antibodies on fixed tissue sections of bovine corpus luteum. The polyclonal antibodies were used successfully in Western blots and specifically reacted with P450scc and 3β‐HSD protein in bovine luteal cell extracts. Thus, P450scc and 3β‐HSD are two specific polyclonal antibodies that are integral products in the investigation of the biological function and regulatory mechanism involved in steroidogenesis.

AMP-activated protein kinase activators in diabetic ulcers: from animal studies to Phase II drugs under investigation

Introduction: Diagnosed cases of diabetes have gradually increased year by year, and research on diabetes mellitus (DM) has attracted greater attention from the medical profession. Diabetic ulcers present persistent pain and the risk of bacterial infection. However, no promising treatment methods have been found. As a regulator of cellular energy balance, 5’ adenosine monophosphate-activated protein kinase (AMPK) has been suggested as a drug target for DM, including such drugs as metformin. Areas covered: This review summarizes the current research and clinical trials of AMPK activators on diabetic wound healing and diabetic ulcers. Furthermore, it discusses the feasibility of AMPK activators in the treatment of diabetic wounds. Expert opinion: Animal studies have demonstrated that AMPK activators are a potential treatment for diabetic ulcers. AMPK activators alleviate tissue inflammation and promote re-epithelialization in diabetic wounds. However, due to the complicated pathological mechanism of diabetic foot ulcers, AMPK activators should be combined with other approaches. The new strategies for combination therapy with AMPK activator may provide a therapeutic advantage for patients with diabetic ulcers.