The Effect and Mechanism of Fructus lycii on Improvement of Exercise Fatigue Using a Network Pharmacological Approach with in vitro Experimental Verification.

Objective: This study aimed to investigate the effect and underlying mechanism of Fructus lycii in improving exercise fatigue. Methods: A network pharmacological approach was used to explore potential mechanisms of action of Fructus lycii. Skeletal muscle C2C12 cells and immunofluorescence were employed to verify the effect and mechanism of the representative components in Fructus lycii predicted by network pharmacological analysis. Results: Six potential active components, namely quercetin, ß-sitosterol, stigmasterol, 7-O-methylluteolin-6-C-beta-glucoside_qt, atropine, and glycitein, were identified to have potency in improving exercise fatigue via multiple pathways, such as the PI3K-Akt, neuroactive ligand-receptor interaction, IL-17, TNF, and MAPK signaling pathways. The immunofluorescence results indicated that quercetin, a significant active component in Fructus lycii, increased the mean staining area of 2-NBDG, TMRM, and MitoTracker, and decreased the area of CellRox compared to the control. Furthermore, the protein expression levels of p-38 MAPK, p-MAPK, p-JNK, p-PI3K, and p-AKT markedly increased after quercetin treatment. Conclusion: Fructus lycii might alleviate exercise fatigue through multiple components and pathways. Among these, quercetin appears to improve exercise fatigue by enhancing energy metabolism and reducing oxidative stress. The PI3K-AKT and MAPK signaling pathways also appear to play a role in this process.

Activation of mitochondrial-associated apoptosis signaling pathway and inhibition of PI3K/Akt/mTOR signaling pathway by voacamine suppress breast cancer progression.

BACKGROUND: Breast cancer is one of the malignant tumors with the highest morbidity and mortality rate. Numerous efficient anti-breast cancer drugs are being derived from the development of natural products. Voacamine (VOA), a bisindole alkaloid isolated from Voacanga africana Stapf, possesses various pharmacological and biological activities. PURPOSE: In this study, we investigated the efficacy of VOA against breast cancer cells and elucidated the underlying mechanisms in vitro and in vivo. METHODS: Human breast cancer cell line MCF-7 and mouse breast cancer cell line 4T1 were used to study the underlying anti-cancer mechanisms of VOA. The proliferation was detected by MTT, colony formation, cell proliferation and wound-healing migration assays. Flow cytometry was utilized to determine the level of reactive oxygen species (ROS) cell-cycle, apoptosis and mitochondrial membrane potential. The target proteins were analyzed by Western blot. Molecular docking was performed and scored by AutoDock. Subcutaneous cancer models in mice were established to evaluate the anticancer effects in vivo. RESULT: Our results demonstrated that VOA selectively suppressed breast cancer MCF-7 and 4T1 cells proliferation with IC50 values of 0.99 and 1.48 µM, and significantly inhibited the migration and colony formation of tumor cells. Furthermore, the cell cycle was arrested in the S phase with the decreased expression levels of CDK2, Cyclin A and Cyclin E. Additionally, exposure to VOA dose-dependently brought about dose-dependently the loss of mitochondrial membrane potential (Δψm) and amassment of reactive oxygen species (ROS), resulting in the initiation of the intrinsic apoptotic pathway. Western blot analysis unveiled that VOA significantly activated mitochondrial-associated apoptosis and obviously suppress the PI3K/Akt/mTOR pathway via modulation of related protein expression levels in both tumor cell lines. In tumor-bearing mouse models, administration of VOA dose-dependently inhibited the tumor growth without causing apparent toxicities. CONCLUSION: These findings revealed the novel properties of VOA in promoting apoptosis of breast cancer cells by activating mitochondrial-associated apoptosis signaling pathway and inhibiting PI3K/Akt/mTOR signaling pathway and significantly decreasing tumor size without detecting appreciable toxicity. In summary, the present results demonstrated VOA could be an encouraging drug candidate to cure breast cancer, exhibiting an effective method to exploit unique drugs from natural components.

A Litopenaeus vannamei TRIM32 gene is involved in oxidative stress response and innate immunity.

Tripartite motif (TRIM) family proteins are named by the presence of tripartite motifs in their amino terminal domains. Apart from the amino terminal, their carboxyl terminal contain variable domains which mediate diverse functions of the TRIM proteins. It had been found that TRIM proteins played important roles in distinct biological processes, such as innate immunity, anti-tumor immunity, cell cycle regulation and so on. In the present study, we cloned a TRIM32 (LvTRIM32) gene from Litopenaeus vannamei. LvTRIM32 was highly expressed in hemocytes, gills and epidermis, and subcellular localization analysis indicated that it was widely distributed in S2 cells. In vitro ubiquitination assays indicated that LvTRIM32 had E3 ubiquitin ligase activity. Results of real-time RT-PCR assay showed that LvTRIM32 was induced in shrimp hemocytes upon oxidative stress. It was also proved that the promoter activity of LvTRIM32 was enhanced by NF-E2-related factor, and knocked-down expression of LvTRIM32 depressed the expression of malic enzyme and epoxide hydrolase. Downregulated LvTRIM32 suppressed the cumulative mortality of shrimp under oxidative stress. Moreover, it was found that LvTRIM32 could be induced in shrimp hemocytes upon immunostimulation, and downregulated LvTRIM32 increased the cumulative mortality of shrimp infected with white spot syndrome virus (WSSV) or Vibrio alginolyticus. Collecting results suggested that LvTRIM32 was a member of shrimp antioxidant stress system, and it was also involved in WSSV- or V. alginolyticus-infection resistance.

Identification and functional characterization of a C-type lectin gene from Litopenaeus vannamei that is associated with ER-stress response.

C-type lectins (CTLs), which bind carbohydrates in a Ca2+-dependent manner, are involved in many cellular activities, especially immunity. CTLs play important roles in both the antibacterial and the antiviral immune response and are also associated with autoimmunity. Several CTLs have been investigated in crustaceans, primarily with respect to their function in the immune response. In this study, we cloned a novel CTL gene (LvCTLU) from Litopenaeus vannamei. LvCTLU is involved in microbe agglutination and phagocytosis. Downregulating LvCTLU increased the cumulative mortality of L. vannamei after Vibrio parahemolyticus infection. Similar to other reported CTLs, LvCTLU also had antiviral properties. Downregulation of LvCTLU also increased the cumulative mortality of L. vannamei after infection with white spot syndrome virus. More importantly, LvCTLU expression was induced by the unfolded protein response (UPR), which is the key pathway in the endoplasmic reticulum (ER)-stress response of eukaryotic organism. Our results suggested that this protein might be involved in the shrimp ER-stress response. Reporter gene assay indicated that LvCTLU was regulated by X-box-binding protein 1, which is the key transcription factor in the UPR. Our study thus revealed that LvCTLU plays vital roles in both the anti-pathogen immune response and the ER-stress response.

Functional characterization of a matrix metalloproteinase 2 gene in Litopenaeus vannamei.

Matrix metalloproteinases (MMPs) contribute to both normal and pathological tissue remodeling. They act as regulatory molecules by working in enzyme cascades as well as processing matrix proteins, cytokines, growth factors and adhesion molecules to generate fragments with biological effects. So MMPs could play distrinct roles in the process of pathogen infection. In present study, we cloned a MMP-2 (LvMMP-2) gene from Litopenaeus vannamei. LvMMP-2, highly expressed in epidermis, located to endoplasmic reticulum in S2 cells. Results of real-time RT-PCR assay showed that LvMMP-2 was induced in shrimp hemocytes upon unfolded protein response or oxidative stress, but not via heat shock treatment. It is proved that the promoter activity of LvMMP-2 was enhanced by NF-E2-related factor 2 and AP-1 factor c-Jun. Further research showed that down-regulated LvMMP-2 contributing to oxidative stress injury, could reduce the cumulative mortality of shrimps under oxidative stress. Besides, our study also indicated that LvMMP-2 was accelerated by lipopolysaccharides injection. LvMMP-2 in S2 could increase the promoter activity of several antimicrobial peptide genes, and knocked-down expression of LvMMP-2 depressed the expression of penaeidin2 and ß-Defensin. Moreover, we showed that down-regulated LvMMP-2 suppressed the cumulative mortality of shrimp infected with white spot syndrome virus (WSSV) or with Vibrio alginolyticus. Collecting results suggested that LvMMP-2 involves in shrimp innate immune response, and also contributes to tissue injury caused by WSSV infection.

Functional characterization of an ER-stress responding Crustin gene in Litopenaeus vannamei.

Shrimp in culture ponds are challenged by various pathogens as well as harsh water environment. The innate immune system and environmental stress response system of shrimp paly an important role in shrimp survival and growth. For remission the endoplasmic reticulum (ER)-stress caused by environmental stress, unfolded protein response (UPR) may reduce the synthesis of most proteins, including great mass of immune factors, which could weaken the immune function of shrimp. Therefore, how cells keep appropriate amount of immune factor synthesis under such a situation is critical important for shrimp health and growth. In this study, we cloned a new Crustin gene (LvCruU) from Litopenaeus vannamei. We showed that LvCruU has antibacterial activity, and reducing its expression would increase the cumulative mortality of L. vannamei upon the Vibrio parahemolyticus infection. In addition, we found that promoter activity of LvCruU was enhanced not only by the deformed epidermal autoregulatory factor-1 (Deaf1), but also by activating transcription factor 3 (LvATF3) of shrimp UPR. Real-time RT-PCR showed that LvCruU and LvATF3 both were induced upon UPR activation. And moreover, in Thapsigargin plus dsLvCruU injection test, we showed that down-regulation of LvCruU increased the cumulative mortality of V. parahemolyticus-infected shrimp under ER-stress. These results suggest that LvCruU work as a downstream effector of UPR, and contribute to antimicrobic immune response upon ER-stress in L. vannamei.

Levels, congener profiles, and dietary intake assessment of polychlorinated dibenzo-p-dioxins/dibenzofurans and dioxin-like polychlorinated biphenyls in beef, freshwater fish, and pork marketed in Guangdong Province, China.

Persistent organic pollutants such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) consisting of non-ortho and mono-ortho PCBs are suggested to be very hazardous and have adverse effects on human health. However, their levels and congener profiles in retail foods marketed in Guangdong Province of China have not been elucidated thus far. Thus, in this study, 226 individual samples of beef, freshwater fish, and pork marketed across four regions of Guangdong Province were randomly collected during 2013-2015 to determine their levels of PCDD/Fs and DL-PCBs. The results showed that the total toxic equivalency quantities (TEQs) of most samples were below the maximum limits except for the 26 samples collected from the vicinities of pollution areas. The median total TEQs of these three categories were 0.174, 0.488, and 0.113pgTEQ/g fw, respectively, which indicated that the contamination status of the studied foods was not serious. For congener profiles, significantly different patterns were observed in three food groups, but with the same major TEQ contributors being 2,3,4,7,8-PeCDF in beef, freshwater fish, and pork. Regional differences of congener profiles in each food group were also found in this study, which might be attributed to the regionally different distributions of PCDD/Fs and DL-PCBs in environment media. The dietary exposures of four population subgroups (girls, boys, male adults, and female adults) to PCDD/Fs and DL-PCBs via three food groups were estimated to assessed the potential risks. They were all lower than the provisional tolerable monthly intake (PTMI, 70pgTEQ/kgbw/month) established by Joint FAO/WHO Expert Committee on Food Additive. In these food categories, the exposure to PCDD/Fs and DL-PCBs via freshwater fish was the highest one, which accounted for about 20% of PTMI, indicating that it was the major route to expose dioxin compounds.

Litopenaeus vannamei activating transcription factor 6 alpha gene involvement in ER-stress response and white spot symptom virus infection.

A previous study found that inositol-requiring enzyme-1-X-box binding protein 1 (IRE1-XBP1) pathway and the protein kinase RNA (PKR)-like ER kinase-eIF2α (PERK-eIF2α) pathway of shrimp play roles in the unfolded protein response (UPR). And they also be proved that was involved in white spot symptom virus (WSSV) infection. Yet the functions of the third branch in shrimp UPR are still unclear. In this study, we showed that upon UPR activation, activating transcription factor 6 alpha (LvATF6α) of Litopenaeus vannamei was cleaved and transferred from the cytoplasm to the nucleus in 293T cells, indicating that the ATF6 pathway in shrimp is also a branch of UPR. Furthermore, LvATF6α could reduce the apoptosis rate of Drosophila Schneider 2 (S2) cells treated with actinomycin, and knock-down expression of LvATF6α increased the apoptosis rate of shrimp hemocytes. In vivo testing revealed that the short from LvATF6α (LvATF6α-s) was obviously increased after UPR activation or WSSV infection, indicating that the ATF6 pathway was activated in L. vannamei gills under such circumstances. Moreover, knock-down expression of LvATF6α could reduce the cumulative mortality and WSSV copy number in WSSV-infected shrimp. Further study revealed that WSSV may profit from shrimp ATF6 pathway activation in two aspects. First, LvATF6α-s significantly upregulated the expression of the WSSV genes (wsv023, wsv045, wsv083, wsv129, wsv222, wsv249, and wsv343). Second, LvATF6α-s inhibited apoptosis by negatively regulating the apoptosis signal-regulating kinase 1 - (c-Jun N-terminal kinase) pathway. All of these evidences suggested that the ATF6 pathway is a member of the L. vannamei UPR, and it is also engaged in WSSV infection.

Quality evaluation of Hypericum ascyron extract by two-dimensional high-performance liquid chromatography coupled with the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.

In this paper, a heart-cutting two-dimensional high-performance liquid chromatography coupled with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was established for controlling the quality of different batches of Hypericum ascyron extract for the first time. In comparison with the common one-dimensional fingerprint, the second-dimensional fingerprint compiled additional spectral data and was hence more informative. The quality of H. ascyron extract was further evaluated by similarity measures and the same results were achieved, the correlation coefficients of the similarity of ten batches of H. ascyron extract were ＞0.99. Furthermore, we also evaluated the quality of the ten batches of H. ascyron extract by antibacterial activity. The result demonstrated that the quality of the ten batches of H. ascyron extract was not significantly different by MTT. Finally, we demonstrated that the second-dimensional fingerprint coupled with the MTT method was a more powerful tool to characterize the quality of samples of batch to batch. Therefore the proposed method could be used to comprehensively conduct the quality control of traditional Chinese medicines.

Isolation and identification of methanethiol-utilizing bacterium CZ05 and its application in bio-trickling filter of biogas.

A bacterium capable of methanethiol (MT) degradation was enriched and isolated by employing activated sewage sludge as the inoculum in a mineral medium containing MT. The isolate was identified as Paenibacillus polymyxa CZ05 through a Biolog test and 16S rDNA sequencing. This strain can utilize both organic and inorganic media and thrives at pH 4 to 9. The batch culture showed that the strain can degrade MT better in the No. 4 medium than in the No. 1 medium. A series-operating biotrickling filter with lava stone as the carrier was employed to test the application of P. polymyxa CZ05 in the removal of MT in simulated biogas. Long-term experiments showed that a high concentration of MT (60 ppm) was efficiently removed (99.5%) by the biotrickling filters at EBRT 30 s. The addition of hydrogen sulfide decreased the MT removal rate because the dissolved oxygen competed with MT.

Cloning of IRAK1 and its upregulation in symptomatic mandarin fish infected with ISKNV.

Interleukin-1 receptor activated kinases (IRAKs) play crucial roles in the Toll-like receptor (TLR) mediated signal transduction pathways that control host innate immune responses. Here we report the cloning of an IRAK1 cDNA (named ScIRAK1) from the mandarin fish. The predicted ScIRAK1 peptide contains a death domain and a serine/threonine-specific kinase domain. Quantitative RT-PCR showed that ScIRAK1 mRNA was primarily expressed in blood cells and posterior kidney. Seven days following infection with infectious spleen and kidney necrosis virus (ISKNV), the ScIRAK1 mRNA level was significantly higher in the blood cells of clinically symptomatic fish than in the blood cells of asymptomatic fish or control fish injected with phosphate-buffered saline. Additional experiments showed that overexpression of ScIRAK1 in the 293T cells could induce NF-kappaB activation. These results suggest that ScIRAK1 may play a role in the pathology of ISKNV infection in the mandarin fish.