Huperzine A attenuates nonalcoholic fatty liver disease by regulating hepatocyte senescence and apoptosis: an in vitro study.

OBJECTIVE: This study was undertaken to detect if free fatty acids (FFA) induce hepatocyte senescence in L-02 cells and if huperzine A has an anti-aging effect in fatty liver cells. METHODS: L-02 cells were treated with a FFA mixture (oleate/palmitate, at 3:0, 2:1, 1:1, 1:2 and 0:3 ratios) at different concentrations. Cell viability and fat accumulation rate were assessed by a Cell Counting Kit 8 and Nile Red staining, respectively. The mixture with the highest cell viability and fat accumulation rate was selected to continue with the following experiment. The L-02 cells were divided into five groups, including the control group, FFA group, FFA + 0.1 µmol/L huperzine A (LH) group, FFA + 1.0 µmol/L huperzine A (MH) group and FFA + 10 µmol/L huperzine A (HH) group, and were cultured for 24 h. The expression of senescence-associated ß-galactosidase (SA-ß-gal) was detected by an SA-ß-gal staining kit. The expression levels of aging genes were measured by qRT-PCR. The expression levels of apoptosis proteins were detected by a Western blot. ELISA kits were used to detect inflammatory factors and oxidative stress products. The expression of nuclear factor (NF-κB) and IκBα were detected by immunofluorescence. RESULTS: The FFA mixture (oleate/palmitate, at a 2:1 ratio) of 0.5 mmol/L had the highest cell viability and fat accumulation rate, which was preferable for establishing an in vitro fatty liver model. The expression of inflammatory factors (TNF-α and IL-6) and oxidants Malonaldehyde (MDA), 4-hydroxynonenal (HNE) and reactive oxygen species (ROS) also increased in the L-02 fatty liver cells. The expression levels of aging markers and aging genes, such as SA-ß-gal, p16, p21, p53 and pRb, increased more in the L-02 fatty liver cells than in the L-02 cells. The total levels of the apoptosis-associated proteins Bcl2, Bax, Bax/Bcl-2, CyCt and cleaved caspase 9 were also upregulated in the L-02 fatty liver cells. All of the above genes and proteins were downregulated in the huperzine A and FFA co-treatment group. In the L-02 fatty liver cells, the expression of IκBα decreased, while the expression of NF-κB increased. After the huperzine A and FFA co-treatment, the expression of IκBα increased, while the expression of NF-κB decreased. CONCLUSION: Fatty liver cells showed an obvious senescence and apoptosis phenomenon. Huperzine A suppressed hepatocyte senescence, and it might exert its anti-aging effect via the NF-κB pathway.

[Combined toxicity of cadmium and S-metolachlor to Scenedesmus obliquus].

The single and combined effects of Cd2+ and S-metolachlor on acute toxicity, total soluble protein content, superoxide dismutase (SOD) activity and cell membrane permeability of Scenedesmus obliquus (S. obliquus) were studied using the standard toxic testing methods. The results showed that the EC50 of Cd2+ and S-metolachlor decreased with time, and the acute toxicity of S-metolachlor was higher than that of Cd2+, EC(50-24h) of Cd2+ and S-metolachlor was 0.27 mg x L(-1) and 0.24 mg x L(-1), respectively, and EC(50.96h) was 0.16 mg x L(-1) and 0.13 mg x L(-1), respectively. The combined toxicity of Cd2+ and S-metolachlor showed a synergistic effects at low concentration, and antagonism effects at high concentration. After 96h-exposure, the total soluble protein content of S. obliquus decreased, the SOD activity first increased and then decreased, and the cell membrane permeability increased with the increasing concentration of both single and combined treatment.

[Influence of S-metolachlor and Cd2+ on photosynthesis of Scenedesmus obliquus].

The single and combined effects of Cd2+ and S-metolachlor on the chlorophyll content and chlorophyll fluorescence parameters of Scenedesmus obliquus were studied by using standard toxic testing method. Both Cd2+ and S-metolachlor had effects on the chlorophyll content and fluorescence parameters such as F0, FV/Fm, FV/F0, Y( II), qP, NPQ and rETR after 96 h-exposure, showing that Cd2+ and S-metolachlor damaged the PS II in algae, inhibited the primary reaction of photosynthesis, stopped the process of photosynthetic electron transport, and destroyed its ability of heat dissipation. The effects of Cd2 + on the chlorophyll content and fluorescence parameters were greater than those of S-metolachlor, and the effects increased with the increasing concentration. The average drop of Y( II ) was 62. 5% in the control group when the light intensity was 231 µmol (m2.s) -1 , and it was 68. 0% , 82. 5% and 100% respectively in Cd2+ -treated groups, and 66. 1% , 72. 1% and 79.6% respectively in S-metolachlor-treated group with the increasing concentration. The combined effects of Cd2+ and S-metolachlor on the chlorophyll fluorescence parameters were mainly due to the impacts of Cd2+.

[Influence of the coexistence of Zn2+ on the enantioselective toxicity of metolachlor to Scenedesmus obliquus].

To evaluate the enantioselective toxicity of chiral pesticide coexisting with heavy metal, the enantioselective toxicity of Rac-, S-metolachlor alone and coexisting with Zn2+ on Scenedesmus obliquus was studied by using standard toxic testing method. The results showed that the trend of the enantioselective toxicity of Rac- and S-metolachlor coexisting with Zn2+ was similar to that of Rac- and S-metolachlor alone. The growth inhibition rate of Scenedesmus obliquus was decreased by the coexistence of Zn2+ with high concentrations of metolachlor. The inhibition rates with 0.30 mg x L(-1) Rac- and S-metolachlor alone at 24 h were 49.61% and 59.73%, and in the coexistence of Zn2+ the values were 38.41% and 42.52%, respectively. The enantioselective toxicity of Rac- and S-metolachlor was expanded and the toxicity of S-metolachlor increased greater than that of Rac-metolachlor. The coexistence of Zn2+ showed partial increase in toxicity of metolachlor in low concentrations, while there was antagonistic effect in high content of metolachlor. The trend of Chlorophyll content of Scenedesmus obliquus at 96 h was in accordance with the growth inhibition.

[Study on extraction and antigenicity of the outer-membrane-protein of Bordetella avium].

To study the immunogenicity of Bordetella avium OMP, ultrasonic dispersion, TritonX-100 technique were used to extract Bordetella avium OMP. Its content was determined by Bradford method and it was detected by SDS-PAGE. Then OMP immunizing antigen was prepared and 1-day chicken was vaccinated by hypodermic inoculation, with the immunizing does of 0.3mL (OMP90microg), 0.5mL (OMP150microg), 0.8mL (OMP240microg), respectively. Results showed that content of Bordetella avium OMP is 300 g/mL, the best immunizing does is 0.5mL each one and Chickens can be protected against Bordetella avium at fetal dose if antibody titer is over 1:2(8). We can see from the antibody level detected by indirect ELISA that antibody can last long enough to help chickens pass susceptible period, so OMP has good immunogenicity. Results of this study lay good foundation for the development of monoclonal antibody to OMP, rapid diagnosis kit and subunit vaccine.