[Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice].

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1ß, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1ß in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1ß, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1ß in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

Phytotoxicity and accumulation of Cu in mature and young leaves of submerged macrophyte Hydrilla verticillata (L.f.) Royle.

Phytotoxicity and accumulation of Cu in mature and young leaves of submerged macrophyte Hydrilla verticillata (L.f.) Royle were investigated by analyzing the chlorophyll contents, chloroplast ultrastructure and leaf surface structure under different Cu treatments (0, 0.01, 0.05 and 0.1 f mg L-1). The results showed that 0.05 and 0.1 mg L-1 Cu treatment decreased the contents of Chl a and Chl b, and caused damage on leaf surface structure and chloroplast ultrastructure compared with control (0 mg L-1 Cu treatment). Higher concentration of Cu induced Chlorophyll decreases and the damages on the leaf surface structure and chloroplast ultrastructure were more pronounced in mature than in young leaves. It was observed that leaf Cu concentration increased almost linearly with exposure time and majority of the Cu accumulated in the cell walls. Among different cell wall fractions, the majority of Cu accumulated in cell walls was bound to the hemicellulose 1 and cellulose, followed by the pectin, hemicellulose 2. Mature leaves had significantly higher the concentrations of total Cu and bound-Cu in cell walls due to higher uronic acid content in their cell wall fractions (pectin, hemicellulose 1 and cellulose) than young leaves. Distinct cell wall composition might partially contribute to the different Cu toxicity and accumulation between mature and young leaves of submerged macrophyte H. verticillata. Our results show that mature leaves are more efficient in the uptake and accumulation of Cu than young leaves, which might explain why mature leaves sustain more severe damage.

Effects of excess ammoniacal nitrogen (NH4+-N) on pigments, photosynthetic rates, chloroplast ultrastructure, proteomics, formation of reactive oxygen species and enzymatic activity in submerged plant Hydrilla verticillata (L.f.) Royle.

Although excess ammoniacal-nitrogen (NH4+-N) results in the disturbance of various important biochemical and physiological processes, a detailed study on the effects of NH4+-N stress on the photosynthesis and global changes in protein levels in submerged macrophytes is still lacking. Here, the changes of excess NH4+-N on physiological parameters in Hydrilla verticillata (L.f.) Royle, a submerged macrophyte were investigated, including the contents of photosynthetic pigments, soluble sugars, net photosynthesis and respiration, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, chloroplast ultrastructure, chloroplast reactive oxygen species (ROS) accumulation and protein levels. Our results showed that the net photosynthetic rate and pigment content reached maximum values when the plants were treated with 1 and 2 mg L-1 NH4+-N, respectively, and decreased at NH4+-N concentrations at 5, 10, 15 and 20 mg L-1. This decrease might be caused by ROS accumulation. Compared that in 0.02 mg L-1 NH4+-N as a control, ROS generation in chloroplasts significantly increased in the presence of more than 2 mg L-1 NH4+-N. Consistently, the damages caused by over-accumulated ROS were observed in chloroplast ultrastructure, showing a loose thylakoid membranes and swollen grana/stroma lamellae. Furthermore, through proteomic analysis, we identified 91 differentially expressed protein spots. Among them, six proteins involved in photosynthesis decreased in abundance in response to excess NH4+-N. Surprisingly, the abundance of all the identified proteins that were involved in nitrogen assimilation and amino acid metabolism tended to increase under excess NH4+-N compared with the control, suggestive of the imbalanced carbon and nitrogen (C-N) metabolisms. In support, activated GS and GOGAT cycle was observed, evidenced by higher activities of GS and GOGAT enzymes. To our knowledge, this work is the first description that excess NH4+-N results in chloroplast ultrastructural damages and the first proteomic evidence to support that excess NH4+-N can lead to a decline in photosynthesis and imbalance of C-N metabolism in submerged macrophytes.

Subcellular accumulation and source of O2- and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle under NH4+-N stress condition.

In this study, the effects of excess NH4+-N on the subcellular accumulation of O2- and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle were investigated using both histochemical and cytochemical methods. Treatments with ≥ 2.00 and ≥ 5.00 mg L-1 NH4+-N for 5 d significantly increased production of O2- and H2O2, respectively. The activities of plasma membrane-bound NADPH (nicotinamide adenine dinucleotide phosphate) oxidases and antioxidant enzymes (superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, dehydroascorbate reductase and glutathione reductase) were also increased correspondingly. This study also provides the first cytochemical evidence of subcellular accumulation of O2- and H2O2 in the submerged plants. In the leaves of H. verticillata treated with 20.0 mg L-1 NH4+-N, O2- dependent DAB precipitates were found primarily on the inner side of the plasma membrane, extracellular space and chloroplasts. H2O2-CeCl3 precipitates were mainly localized on the inner side of the plasma membrane and extracellular space of the mesophyll cells. Treatments with the inhibitors of NADPH oxidase (diphenylene iodonium and imidazole) indicate that NH4+-N-induced production of O2- and H2O2 in H. verticillata leaves may involve plasma membrane-bound NADPH oxidase. Moreover, low-light treatment decreased NH4+-induced O2- production, suggesting that alterations in the photosynthetic electron transfer chain due to NH4+ toxicity could lead to O2- production.

Hydrilla verticillata employs two different ways to affect DNA methylation under excess copper stress.

Because of the accumulation of heavy metals, Hydrilla verticillata (L.f.) Royle, a rooted submerged perennial aquatic herb, is being developed as a potential tool to clean the aquatic ecosystem polluted by heavy metals. However, its physiological responses for heavy metal remain to be elucidated. Here, through employing proteomics approach, we found that excess Cu significantly induced the expressions of four DNA methylation related proteins in H. verticillata, which were the homologues of two domains rearranged methyltransferases (DRM), a methyltransferases chromomethylase (CMT) and a histone H3 lysine-9 specific SUVH6-like (SUVH6). Consistently, a dramatic change in DNA methylation patterns was detected in excess Cu-exposed H. verticillata. Surprisingly, administration of the NADPH oxidase inhibitors, diphenylene iodonium (DPI) and imidazole (IMZ) that block production of reactive oxygen species (ROS) could trigger the remethylation of genomic sites that were demethylated by excess Cu, indicating that Cu-induced ROS might be another way to affect DNA methylation. Further analysis suggested this changed DNA methylation may be owing to the ROS-induced DNA damage. Taken together, our findings demonstrate that two different ways to influence DNA methylation in excess Cu-treated H. verticillata.