DADS Inhibits the Proliferation of MDCC-MSB-1 Cells by Inducing Autophagy via the MEK/ERK Signalling Pathway.

Diallyl disulfide (DADS) is effective at suppressing tumour cell growth and proliferation. This study verified the morphology and growth activity of MDCC-MSB-1 cells by using an MTT assay to detect the effect of DADS on the proliferation of MDCC-MSB-1 cells and a CCK8 assay to detect the effect of DADS on the viability and proliferation of MDCC-MSB-1 cells. We found that the viability and proliferation of MDCC-MSB-1 cells decreased with increasing DADS concentrations. MDC staining and Western blotting were used to analyse autophagy, the associated protein LC3 and the MEK/ERK pathway proteins MEK and ERK and to investigate changes in cellular autophagy based on cell morphology and molecular biology. With increasing concentrations of DADS, MDCC-MSB-1 cell autophagy increased in a gradient manner. Additionally, the conversion of the autophagy marker protein LC3-I increased with increasing drug concentrations, and the relative expression of LC3-II steadily increased, as did the expression of key protein components of the MEK/ERK signalling pathway, including P-MEK1/2 and P-ERK1/2. These results suggest that DADS induces autophagy through the MEK/ERK pathway, thereby inhibiting the proliferation of MDCC-MSB-1 cells.

Houttuynia cordata Thunb repairs steroid-induced avascular necrosis of the femoral head through regulating NF-κB signaling pathway.

To investigate the influences of Houttuynia cordata Thunb (HCT) in steroid-induced avascular necrosis of the femoral head (SANFH), we conducted a comprehensive study evaluating the effects of HCT on various aspects. Cell Counting Kit-8 assay was used to examine bone marrow stem cells (BMSCs) cell viability. Flow cytometry and lactate dehydrogenase detection assay were conducted to determine cell apoptosis. The levels of apoptosis-related proteins, osteogenic-related markers, inflammatory factors, and nuclear factor kappa B (NF-κB) pathway-associated proteins were determined via western blotting. Hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays were utilized to verify the effects of HCT in SANFH rats. Our findings revealed that HCT could enhanced cell viability and arrested cell apoptosis in dexamethasone (Dex)-treated BMSCs. Dex increased the levels of cleaved caspase-3, Bcl2-associated X, interleukin (IL)-1ß, IL-18, IL-6, p65, and inhibitor of NF-κB kinase ß (IKKß), while this promoting trend was weakened by HCT. Moreover, pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB signaling pathway) further increased the inhibitory role of apoptosis and the levels of IL-1ß, IL-18, and IL-6 and the promotional effect of the levels of RUNX2 and ALP in Dex-treated BMSCs. The in-vivo assays showed that HCT decreased the percentage of empty lacunae, apoptosis, and the levels of IL-1ß, IL-18, IL-6, p65, and IKKß in SANFH rats. In conclusion, our study demonstrated that HCT relieved SANFH, which might be possibly achieved by NF-κB pathway.

CRISPR-activation screen identified potassium channels for protection against mycotoxins through cell cycle progression and mitochondrial function.

Zearalenone (ZEA) exposure has carcinogenic effects on human and animal health by exhibiting intestinal, hepatic, and renal toxicity. At present, the underlying mechanisms on how ZEA induces apoptosis and damage to tissues still remain unclear. In this study, we aimed to identify genes that modulate the cellular response to ZEA using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening, and further validate novel gene functions to elucidate molecular mechanisms underlying particular biological processes in vivo and in vitro. Two ZEA-resistant cell lines, designated Ov-KCNJ4 and Ov-KCNJ12, were yielded by CRISPR activation screening which had significant changes in ZEA resistance and growth rates. Results showed that ZEA could interact with the cell membrane proteins KCNJ4 and KCNJ12, inducing cell cycle arrest, disruption of DNA replication and base excision repair. Overexpression of KCNJ4 and KCNJ12 was involved in ZEA resistance by regulating cell cycle to neutralize toxicity, sustaining mitochondrial morphology and function via attenuating the damage from oxidative stress in the KCNJ4-mitoKATP pathway. In vivo experiments showed that AAV-KCNJ4 delivery significantly improved ZEA-induced renal impairment and increased antioxidative enzyme activity by improving mitochondrial function. Our findings suggest that increasing potassium channel levels may be a putative therapeutic target for mycotoxin-induced damage.

Thioredoxin A of Streptococcus suis Serotype 2 Contributes to Virulence by Inhibiting the Expression of Pentraxin 3 to Promote Survival Within Macrophages.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that can infect humans in contact with infected pigs or their byproducts. It can employ different types of genes to defend against oxidative stress and ensure its survival. The thioredoxin (Trx) system is a key antioxidant system that contributes adversity adaptation and pathogenicity. SS2 has been shown to encode putative thioredoxin genes, but the biological roles, coding sequence, and underlying mechanisms remains uncharacterized. Here, we demonstrated that SSU05_0237-ORF, from a clinical SS2 strain, ZJ081101, encodes a protein of 104 amino acids with a canonical CGPC active motif and an identity 70-85% similar to the thioredoxin A (TrxA) in other microorganisms. Recombinant TrxA efficiently catalyzed the thiol-disulfide oxidoreduction of insulin. The deletion of TrxA led to a significantly slow growth and markedly compromised tolerance of the pathogen to temperature stress, as well as impaired adhesion ability to pig intestinal epithelial cells (IPEC-J2). However, it was not involved in H2O2 and paraquat-induced oxidative stress. Compared with the wild-type strain, the ΔTrxA strain was more susceptible to killing by macrophages through increasing NO production. Treatment with TrxA mutant strain also significantly attenuated cytotoxic effects on RAW 264.7 cells by inhibiting inflammatory response and apoptosis. Knockdown of pentraxin 3 in RAW 264.7 cells was more vulnerable to phagocytic activity, and TrxA promoted SS2 survival in phagocytic cells depending on pentraxin 3 activity compared with the wild-type strain. Moreover, a co-inoculation experiment in mice revealed that TrxA mutant strain is far more easily cleared from the body than the wild type strain in the period from 8-24 h, and exhibits significantly attenuated oxidative stress and liver injury. In summary, we reveal the important role of TrxA in the pathogenesis of SS2.

SPRY1 promotes cell proliferation and inhibits apoptosis by activating Hedgehog pathway in acute myeloid leukemia.

OBJECTIVE: The aim of this study was to determine the biological function of Sprouty 1 (SPRY1) on acute myeloid leukemia (AML), and to investigate the potential mechanism. METHODS: The expression of SPRY1 and the prognostic values of SPRY1 were assessed through the analysis of the Cancer Genome Atlas. Meanwhile, the expression of SPRY1 in AML cells was determined by qRT-PCR and western blot. Then, the biological function of SPRY1 on the proliferation, cell cycle and apoptosis in K-562 and HL-60 cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-formation assay, 5-ethynyl-20-deoxyuridine assay and flow cytometry. Additionally, the protein expressions were measured by western blot. RESULTS: We found that SPRY1 was markedly overexpressed in the cells of the patients with AML, and the patients with AML having a high SPRY1 expression has a bad prognosis. The proliferation and cell cycle progression in K-562 and HL-60 cells were notably promoted by SPRY1 overexpression, but inhibited by SPRY1 knockdown. Meanwhile, the apoptosis of K-562 and HL-60 cells was significantly repressed by SPRY1 overexpression and facilitated by SPRY1 knockdown. In addition, we found that SPRY1 overexpression significantly activated the Hedgehog pathway in AML cells. The function of SPRY1 on the proliferation, cell cycle and apoptosis was reversed by Gli1 in K-562 and HL-60 cells. DISCUSSION: Identifying new biomarkers and exploring the pathogenesis of AML is urgent to improve the disease surveillance for patients with AML. CONCLUSIONS: SPRY1 could facilitate cell proliferation and cell cycle progression, and suppress cell apoptosis via activating the Hedgehog pathway in AML.

Factors Associated with Dog Rabies Immunization in Changsha, China: Results of a Cross-Sectional Cluster Survey, 2015-2021.

The objective of this study was to examine longitudinal trends in the prevalence of dogs that are successfully immunized against rabies virus (as measured by sufficient serum antibodies) in Changsha, an urban center of China. The secondary objective was to investigate the factors affecting the seroprevalence of rabies virus antibodies in dogs. In this study, 4515 canine serum samples were collected from 57 pet hospitals (immunization points) during the period of 2015-2021 in five major urban areas of Kaifu, Furong, Tianxin, Yuhua, and Yuelu in Changsha, China. The enzyme-linked immunosorbent assay (ELISA) method was used to analyze the level and trend of rabies virus antibodies in serum and further evaluate the potential factors affecting the immunization effect from five factors: sex, age, time interval after most recent vaccination and sample collection, number of vaccinations, and vaccine manufacturer. The results showed that the seroconversion from the urban dog in Changsha steadily increased from 46.13% to 73.38% during 2015-2017. The seropositivity prevalence remained above the international standard (70%) from 2018 to 2020 and up to 90.99% in 2021. Further analysis showed that the seroconversion of rabies virus among dogs was significantly affected by the age, the number of vaccinations, time interval after the most recent vaccination and sample collection, and vaccine manufacturer, while sex had less influence. The overall rabies vaccination situation in urban areas of Changsha generally meets international standards, with only a few areas showing low levels of antibodies in dogs after vaccination and risk of infectiousness. Therefore, it is recommended that the first vaccination should be given when the dog is about three months old and regularly repeated every year after that. At the same time, antibody concentrations in dogs, especially in newborn puppies and older dogs, need to be tested promptly after vaccination at the required time to ensure that they are at a high level of immune protection, which can strengthen the supervision of rabies.

Synthesis, identification and anti-cancer activity of 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2-enyl)disulfane.

In this study, we synthesized 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2- enyl)disulfane using sodium sulfide, 1-bromine-4-methyl-2-amylene and 1-(4-bromine-2- butylene)benzene as raw materials. The yield rate of target product was 84%. The structure of the target product was confirmed by GC-MS, 1H-NMR and elemental analysis. The results of anti-cancer activity experiments showed that 1-(4-methylpent-2-enyl)-2-(4- phenylbut-2-enyl)disulfane could significantly inhibit the proliferation, induce the apoptosis of CNE2 cells in a dose dependent manner, and could significantly enhance the activity of XIAP.

Synthesis and effect on human HepG2 cells of 1,2-bis-(2-methylallyl)disulfane.

1,2-bis(2-methylallyl)disulfane was synthesized from sodium sulfide and 3-chloro-2-methylpropylene. The structure of the target product was confirmed by GC-MS, 1H-NMR and elemental analysis. Cell viability assay, flow-cytometric analysis and protein expression results showed that 1,2-bis(2-methylallyl)disulfane could significantly inhibit the proliferation, and induce the apoptosis of human HepG2 cells.

The roles of p38MAPK and caspase-3 in DADS-induced apoptosis in human HepG2 cells.

OBJECTIVES: To explore the function of p38MAPK and caspase-3 in DADS-induced apoptosis in human HepG2 cells, and discuss the signal transduetion mechanism of HepG2 cells in the apoptosis process induced by DADS by using the inhibitors of p38MAPK (SB203580) and caspase-3 (Z-DEVD-FMK). METHODS: After the human HepG2 cells had been treated with the DADS and inhibitors for 24 h, cell viability was determined by the MTT method, apoptosis was evaluated by flow cytometry (FCM) and the expressions of p38MAPK and caspase-3 were measured by western-blot. RESULTS: Our results indicated that DADS activities the p38MAPK and caspase-3, but the inhibitors, SB203580 and Z-DEVD-FMK (for p38MAPKand for caspase-3, respectively), both have the effect of inhibitory activity on P38MAPK and caspase-3. Furthermore, a combination treatment with both DADS and inhibitor (SB203580 or Z-DEVD-FMK) decreases the inhibitory and apoptotic activity of HepG2 cells increased compared with DADS-treated. CONCLUSIONS: Our data indicate that p38MAPK and caspase-3 are involved in the process of DADS-induced apoptosis in human HepG2 cells and interact with each other.