Effects Of Some Important Exogenous Factors On Cell Viability, Oxidative Damage, Apoptosis And Heat Shock Protein Expression In Hepatic Cells Of Grass Carp (Ctenopharyngodon Idellus)

The grass carp (Ctenopharyngodon idellus) is a popular freshwater fish in China because of its rapid growth, high yield, and low-cost production. It therefore plays an important role in aquaculture, with a total production of 4.54 million tons in 2011, which was the highest in the world (FAO,2011). This fish has been introduced as a food fish or a biological controller of aquatic vegetation into more than 100 countries in various continental regions, including East Asia, North America, and Europe. However, grass carp cultured in China as well as in other parts of the world have suffered from serious disease problems, drug and stress.For these reasons, the first study was to determine the effects of three different hyperthermia treatments and recovery from them and stress time on cell viability oxidative damage, and heat shock protein expression in the hepatic of grass carp. In addition, we examined effects the effect of emodin and enrofloxacin on cytotoxicity, apoptosis and antioxidant capacity in the hepatic cells of grass carp. At last, we analyzed protective effects of emodin and pre-heating against further stress in the hepatic cells of grass carp. This study would provide new ideas for anti-stress of grass carp further.1. Effect of heat stress and recovery on viability, oxidative damage, and heat shock protein expression in hepatic cells of grass carpIn this study, we investigated the effects of hyperthermia and recovery on cell viability, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and heat shock protein (HSP60,70, and 90) mRNA expression in the hepatic cells of the grass carp, Ctenopharyngodon idellus. Triplicate groups of cultured cells were exposed to 30℃, 32℃, or 34℃ for 0.5 h and then immediately incubated at 27℃ in 5% CO2 for 6,12,24, or 48 h. Hyperthermia stress greatly reduced cell viability and increased LDH release. Cell damage declined after recovery. Hyperthermia stress increased the lipid peroxide levels and reduced the antioxidant capacity (e.g., reduced SOD and T-AOC) of the cells. However, oxidative damage declined as the recovery period increased, and the levels of MDA, SOD, and T-AOC were restored. After cells were exposed to 32℃, the expression of HSP60 after recovery for 6 and 24 h (P< 0.05), the expression of HSP70 after recovery for 6,24 and 48 h (P< 0.05), and the expression of HSP90 after recovery for 6 and 24 h was significantly higher (P< 0.05) than the prestress levels. During the recovery period, the variations in HSP gene expression reflected the transition period from a state of cellular growth to one of cellular repair. In conclusion, hyperthermia depresses cell viability, induces oxidative damage, and increases HSP expression, which plays an important role during hyperthermic stress in grass carp hepatic cells.2. The effect of hyperthermia on cell viability, oxidative damage, and heat shock protein expression in hepatic cells of grass carpThe purpose of the study was to investigate the effects of mild hyperthermia on cell viability, release of LDH, SOD activity, MDA formation, T-AOC, and the relative mRNA levels of HSPs (HSP60,70, and 90) in hepatic cells of grass carp before and after temperature stress. Cultured cells were exposed to thermal stress (32℃) for 0.5,1,2,4, and 8 h. The results showed that hyperthermia stress significantly reduced cell viability (P< 0.01) and increased LDH release at 0.5 and 1 h (P< 0.05). Additionally, hyperthermia stress led to oxidative stress as evidenced by significantly decreased T-AOC after treating cells for 0.5 and 8 (P< 0.05). SOD activity also significantly decreased after 1 h of stress (P< 0.05), but MDA formation increased after 8 h of stress (P< 0.05). This may be partly responsible for the lower cell viability and higher LDH release we observed. The differences between SOD activity, MDA formation, and T-AOC between the 2 h treatment group and the control were smaller than that of other groups. This indicated that cellular antioxidant enzyme systems play an important role in the defense against oxidative stress. Further tests showed that the expression of HSP60 at 1,2, and 4 h (P< 0.05), HSP70 at 0.5 and 1 h(P<0.01), and HSP90 at all time points after stress were higher (P<0.01) than prestress levels. This suggested that HSPs possess the ability to modulate cellular anti-stress responses and play key roles in protecting organisms from heat stress. In conclusion, hyperthermia inhibits cell proliferation, induces cell oxidative stress, and enhances HSP expression in hepatic cells of grass carp.3. The effect of emodin and enrofloxacin on cytotoxicity, apoptosis, antioxidant capacity and heat shock proteins expression in the hepatic cells of grass carpWe determined the effect of emodin and enrofloxacin on cell viability, LDH release, SOD, glutathione (GSH), T-AOC, reactive oxygen species (ROS), mitochondria membrane potential (△ψm), apoptosis and the relative mRNA levels of HSPs (HSP60,70, and 90) in the hepatic cells of grass carp. Cultured cells were treated with different concentrations of emodin (0.04,0.2,1,5,25 μg·mL-1) and enrofloxacin (12.5,25,50,100, 200 μg·mL-1) for 24 h. We found that the cytotoxic effect of emodin was mediated by apoptosis, and that this apoptosis occurred in a dose-dependent manner. The emodin-induced apoptosis was accompanied △ψm disruption and ROS generation. SOD and T-AOC significantly decreased in the emodin-exposed cells. Thus, the oxidative effect of emodin may be attributed to the loss of the cell’s ability to maintain the activity of its radical-scavenging enzymes. GSH was also significantly higher after emodin exposure, indicating that cells failed to maintain their redox balance when compensating for the increased oxidative stress. HSPs (HSP60,70,90) mRNA levels significantly increased in the groups exposed to 5 and 25 μg·mL-1 of emodin to protect against damage. Furthermore, when cells were exposed to graded levels of enrofloxacin, cell viability significantly reduced and LDH release increased (P< 0.05). Additionally, enrofloxacin induced apoptosis and reduced △ψm. ROS and MDA significantly increased but T-AOC decreased in the enrofloxacin-exposed cells (P< 0.05), which indicated that enrofloxacin can induce lipid peroxidation, lead to oxidative stress. HSP60 and HSP70 expressions were inhibited in the high concentration groups. In addition, the HSP90 mRNA levels in the treatment exposed to 100 and 200 μg·mL-1 enrofloxacin were significantly lower than those of the control group (P< 0.05). It suggested high concentrations enrofloxacin decrease HSPs can lead to pathological damage and inhibition of protein synthesis in the cell.4. Protective effects of prior heat stress against emodin-induced damage in the hepatic cells of grass carpThe aim of the study was to investigate the effects of mild hyperthermia on cell viability, release of LDH, SOD activity, ROS, A^m, and the relative mRNA levels of HSPs (HSP60,70, and 90) against emodin-induced damage in the hepatic cells of grass carp. Triplicate groups of cultured cells were exposed to 30℃,32℃, or 34℃ for 0.5 h and then immediately incubated at 27℃ in 5%　CO2 for 12 h. After that cells were treated with different concentrations of emodin (0.04,0.2,1,5,·25 μg·mL-1) for 24 h. We found that compared to high concentration groups (1,5,25 μgmL-1),30℃ prior heat stress in L8824 significantly increased cell viability (P< 0.05) and reduced content of LDH release (P< 0.05), increased △ψm (P< 0.05), which indicated that 30℃ prior heat stress can reduce the damage induced by emodin; ROS reached a minimum in 30℃ group and was significant lower than emodin-treated groups (P< 0.05), while SOD and T-AOC in 30℃ group were significant higher than emodin-treated groups (P< 0.05), suggested 30℃ mild prior heat stress induce antioxidant enzymes detoxify harmful ROS generation; Further test showed the expression of levels of HSPs enhanced by treating high temperature (30℃,32℃ and 34℃), suggested prior heat stress induce HSPs which inhibit cell and protect L8824 from high concentration emodin injury. In conclusion, prior heat stress has the protective effect of L8824 under emodin and its mechanism may be related with increasing SOD activity, enhancing the HSPs expression and stabilizing the cell membrane potential.5. Protective effects of emodin against hyperthermia-induced stress in the hepatic cells of grass carpWe determined the effect of emodin on cell viability, release of LDH, SOD activity, ROS, △ψm, and the relative mRNA levels of HSPs (HSP60,70, and 90) against hyperthermia-induced stress in the hepatic cells of grass carp. Cultured cells were treated with different concentrations of emodin (0.04,0.2,1 μg·mL-1) for 24 h and then exposed to thermal stress (32℃) for 0.5 h. The results showed that 0.04 μg·mL-1 emodin significantly increased cell viability (P< 0.05) and reduced content of LDH release (P< 0.05), compared to hyperthermia group, which indicated that 0.04 μg·mL-1 emodin can protect the L8824 from hyperthermia injury. Besides, compared to hyperthermia group, 0.04 μg·mL-1 emodin led significant increase of SOD (P< 0.05), but decrease of ROS after stress, which suggested that emodin can increase their antioxidant potential and inhibit ROS generated to protect cell from oxidative stress.0.04 μg·mL-1 emodin increased HSP90 expression may detoxify the ROS generated, inhibit apoptosis and play roles in protecting L8824 from heat stress. In conclusion,0.04 μg·mL-1 emodin has the protective effect on L8824 against cellular damage induced by stress.