The liver is the largest and primary target organ responsible for metabolism and detoxification of drugs and environmental toxicants. This organ also performs a major role on the glucose metabolism, hormone production, decomposition of red blood cells, synthesis of serum protein and production of bi...
The liver is the largest and primary target organ responsible for metabolism and detoxification of drugs and environmental toxicants. This organ also performs a major role on the glucose metabolism, hormone production, decomposition of red blood cells, synthesis of serum protein and production of bile. The biological ability of liver can be declined by the hepatic damage, necrosis and apoptosis induced with many toxicants such as carbon tetrachloride (CCl4) and aflatoxin. Also, various principal causes including virus infection, alcoholic and nonalcoholic fat liver disease (steatohepatitis), autoimmune disorder, cholestatic disorders, and some metabolic diseases induce hepatic diseases. Meanwhile, several natural products have recently received attention as key sources of antioxidants because they are highly active in the prevention and treatment of diseases induced by oxidative stress. Among these products, Galla rhois (GR) is the term used for the gall caused by the Chinese aphid, Schlechtendalia chinensis Bell, on the leaves of Rhus chinensis Mill. GR have exhibited good antibacterial, antioxidant, and anticancer activities. However, more studies are necessary to determine the novel functions and mechanisms of GR in apoptosis of hepatocytes and hepatic disease. Therefore, in this study, we investigated the precautionary effects of GR on hydrogen peroxide (H2O2)-induced apoptosis in HepG2 cell and CCl4-induced hepatic disease of ICR mice.
First of all, in order to investigate whether or not gallotannin-enriched GR (GEGR) could protect against hepatotoxicity induced by H2O2 treatment, cell viability, apoptosis-related proteins expression, and reactive oxygen species (ROS) generation were measured in four different concentrations of GEGR (6.25 ? 50 μg/ml) pretreated HepG2 cells for 24 h before H2O2 exposure. GEGR contained high concentrations of three bioactive components related to its hepatoprotective effects, gallotannin (69.2%), gallic acid (26.6%) and methylgallate (4.2%). GEGR showed remarkable DPPH scavenging activity (IC50=0.212 μg/ml). The LD50 value calculated for the response of HepG2 cells was determined as 178 μg/ml using cell viability assay. Also, GEGR showed outstanding anti-apoptosis effect (ED50=6.85 μg/ml) in H2O2 exposed HepG2 cell. Significant reduction of the immunofluorence intensity indicating apoptosis was also detected in the nuclei of HepG2 cells stained with DAPI and Annexin V after GEGR treatments. The Bax/Bcl-2 ratio and active caspase-3 levels were decreased in GEGR pretreated cells relative to H2O2-Vehicle treated group, even though little or no decrease was observed in response to low GEGR concentrations. Furthermore, the GEGR pretreated group showed a reduced level of DCFH-DA stained cells, representing ROS generation relative to the H2O2+Vehicle treated group.
Second, to investigate the protective effects of GEGR against CCl4-induced acute hepatotoxicity in ICR mice, alterations in serum biochemical indicators, histopathological structure, hepatic apoptosis, liver fibrosis and antioxidative status were measured in ICR mice pretreated with GEGR for 5 days before CCl4 injection. The GEGR/CCl4 treated group showed decreased levels of three serum marker enzymes for liver functionality (ALP, AST and ALT) and necrotic area, while LDH levels remained constant. The level of active caspase-3 and Bax/Bcl-2 expression was effectively inhibited in the GEGR/CCl4 treated group. Moreover, the level of pro-inflammatory cytokines, TNF-α and IL-6, was rapidly recovered in the GEGR/CCl4 treated group, while anti-inflammatory cytokine (IL-10) increased slightly in the same group. Also, the phosphorylation levels of p38 and JNK, a downstream signaling pathway of TNF-α, were recovered to normal in GEGR/CCl4 treated group. Furthermore, the GEGR/CCl4 treated group showed a significant decrease in collagen accumulation, MMP-2 expression, tumor growth factor (TGF)-β1 concentration and the phosphorylation of Smad2/3. Also, a significant decline in malondialdehyde (MDA) concentration and enhancement of superoxide dismutase (SOD) expression was observed in the GEGR preadministrated group.
To evaluate the hepatotoxicity and nephrotoxicity of GEGR toward the liver and kidney of ICR mice, alterations in related markers including body weight, organ weight, urine composition, and histological changes of the liver and kidney were analyzed after oral administration of 250, 500 and 1,000 mg/kg body weight/day of GEGR for 14 days. The concentration of MDA, a marker of lipid peroxidation, was decreased about 19% in the serum of HGEGR-treated mice. No significant alteration was observed in body and organ weight, clinical phenotypes, urine parameters and mice mortality in GEGR treated groups. Also, GEGR administration did not alter level of serum marker enzyme for liver functionality (ALP, ALT, AST, LDH, BUN, and CRE) compared to vehicle treated group. Moreover, the specific pathological features generally induced by toxic compounds were not observed upon liver and kidney histological analysis.
Taken together, these results suggest that GEGR could prevent cell death induced by H2O2 in HepG2 cells via antioxidant activity. Also, GEGR administration effectively suppressed the symptoms of liver injury induced by CCl4 exposure. Moreover, GEGR did not induce any specific toxicity in various organs including liver and kidney of ICR mice. Therefore, we suggest that GEGR can be considered as a beneficial natural compound against liver diseases.
The liver is the largest and primary target organ responsible for metabolism and detoxification of drugs and environmental toxicants. This organ also performs a major role on the glucose metabolism, hormone production, decomposition of red blood cells, synthesis of serum protein and production of bile. The biological ability of liver can be declined by the hepatic damage, necrosis and apoptosis induced with many toxicants such as carbon tetrachloride (CCl4) and aflatoxin. Also, various principal causes including virus infection, alcoholic and nonalcoholic fat liver disease (steatohepatitis), autoimmune disorder, cholestatic disorders, and some metabolic diseases induce hepatic diseases. Meanwhile, several natural products have recently received attention as key sources of antioxidants because they are highly active in the prevention and treatment of diseases induced by oxidative stress. Among these products, Galla rhois (GR) is the term used for the gall caused by the Chinese aphid, Schlechtendalia chinensis Bell, on the leaves of Rhus chinensis Mill. GR have exhibited good antibacterial, antioxidant, and anticancer activities. However, more studies are necessary to determine the novel functions and mechanisms of GR in apoptosis of hepatocytes and hepatic disease. Therefore, in this study, we investigated the precautionary effects of GR on hydrogen peroxide (H2O2)-induced apoptosis in HepG2 cell and CCl4-induced hepatic disease of ICR mice.
First of all, in order to investigate whether or not gallotannin-enriched GR (GEGR) could protect against hepatotoxicity induced by H2O2 treatment, cell viability, apoptosis-related proteins expression, and reactive oxygen species (ROS) generation were measured in four different concentrations of GEGR (6.25 ? 50 μg/ml) pretreated HepG2 cells for 24 h before H2O2 exposure. GEGR contained high concentrations of three bioactive components related to its hepatoprotective effects, gallotannin (69.2%), gallic acid (26.6%) and methylgallate (4.2%). GEGR showed remarkable DPPH scavenging activity (IC50=0.212 μg/ml). The LD50 value calculated for the response of HepG2 cells was determined as 178 μg/ml using cell viability assay. Also, GEGR showed outstanding anti-apoptosis effect (ED50=6.85 μg/ml) in H2O2 exposed HepG2 cell. Significant reduction of the immunofluorence intensity indicating apoptosis was also detected in the nuclei of HepG2 cells stained with DAPI and Annexin V after GEGR treatments. The Bax/Bcl-2 ratio and active caspase-3 levels were decreased in GEGR pretreated cells relative to H2O2-Vehicle treated group, even though little or no decrease was observed in response to low GEGR concentrations. Furthermore, the GEGR pretreated group showed a reduced level of DCFH-DA stained cells, representing ROS generation relative to the H2O2+Vehicle treated group.
Second, to investigate the protective effects of GEGR against CCl4-induced acute hepatotoxicity in ICR mice, alterations in serum biochemical indicators, histopathological structure, hepatic apoptosis, liver fibrosis and antioxidative status were measured in ICR mice pretreated with GEGR for 5 days before CCl4 injection. The GEGR/CCl4 treated group showed decreased levels of three serum marker enzymes for liver functionality (ALP, AST and ALT) and necrotic area, while LDH levels remained constant. The level of active caspase-3 and Bax/Bcl-2 expression was effectively inhibited in the GEGR/CCl4 treated group. Moreover, the level of pro-inflammatory cytokines, TNF-α and IL-6, was rapidly recovered in the GEGR/CCl4 treated group, while anti-inflammatory cytokine (IL-10) increased slightly in the same group. Also, the phosphorylation levels of p38 and JNK, a downstream signaling pathway of TNF-α, were recovered to normal in GEGR/CCl4 treated group. Furthermore, the GEGR/CCl4 treated group showed a significant decrease in collagen accumulation, MMP-2 expression, tumor growth factor (TGF)-β1 concentration and the phosphorylation of Smad2/3. Also, a significant decline in malondialdehyde (MDA) concentration and enhancement of superoxide dismutase (SOD) expression was observed in the GEGR preadministrated group.
To evaluate the hepatotoxicity and nephrotoxicity of GEGR toward the liver and kidney of ICR mice, alterations in related markers including body weight, organ weight, urine composition, and histological changes of the liver and kidney were analyzed after oral administration of 250, 500 and 1,000 mg/kg body weight/day of GEGR for 14 days. The concentration of MDA, a marker of lipid peroxidation, was decreased about 19% in the serum of HGEGR-treated mice. No significant alteration was observed in body and organ weight, clinical phenotypes, urine parameters and mice mortality in GEGR treated groups. Also, GEGR administration did not alter level of serum marker enzyme for liver functionality (ALP, ALT, AST, LDH, BUN, and CRE) compared to vehicle treated group. Moreover, the specific pathological features generally induced by toxic compounds were not observed upon liver and kidney histological analysis.
Taken together, these results suggest that GEGR could prevent cell death induced by H2O2 in HepG2 cells via antioxidant activity. Also, GEGR administration effectively suppressed the symptoms of liver injury induced by CCl4 exposure. Moreover, GEGR did not induce any specific toxicity in various organs including liver and kidney of ICR mice. Therefore, we suggest that GEGR can be considered as a beneficial natural compound against liver diseases.
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