Salidroside Inhibits α-Amanitin-Induced AML-12 Cell Apoptosis via the Regulation of PINK1/Parkin-Mediated Mitophagy and Mitochondrial Function.

Poisoning caused by the mushroom Amanita phalloides, due to the toxin α-amanitin, accounts for approximately 90% of food poisoning deaths in China with no specific antidotes. To investigate the role of salidroside (Sal) in α-amanitin (α-AMA)-induced mitophagy, mouse liver cells AML-12 were exposed to α-AMA in the presence of Sal or not. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit, mitochondrial activity was evaluated using a mitochondrial red fluorescent probe kit or JC-1 dye, and protein expression levels of PINK1, Parkin, LC3 II, P62, Bax, Bcl-2, Caspase 3, Cleaved-Caspase 3, PARP I, and Cleaved-PARP I were detected through Western blot. Results demonstrated that α-AMA led to increased intracellular ROS levels, cell apoptosis, and decreased mitochondrial membrane potential. Notably, expression levels of mitophagy-related proteins PINK1, Parkin, and LC3 increased significantly while the P62 protein expression decreased remarkably. Furthermore, Sal reversed the α-AMA-induced decrease in cell viability and mitochondrial membrane potential and increase in intracellular ROS level. In addition, Sal promoted expression levels of PINK1, Parkin, and LC3 II while suppressing the Bax/Bcl-2 ratio, Cleaved-Caspase 3, and Cleaved-PARP I as well as P62. The results above proved that salidroside alleviates α-AMA-induced mouse liver cells damage via promoting PINK1/Parkin-mediated mitophagy and reducing cell apoptosis.

Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis.

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Fungi, including Pithomyces chartarum cause facial eczema and inflammation in grazing sheep in Western China.

Facial eczema is often found in flocks of grazing sheep in China. To investigate fungi species those cause disease and pathological roles. Forage and soil samples were collected during the pathogenic season and cultured. Samples were collected from regions with and without facial eczema affected sheep. Fungal isolation and identification, statistical analysis of fungal species and distribution were performed. Pathological changes, biochemical parameters of serum liver function and protection of inflammatory factors that tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12) were observed. Fungal cultivation and identification showed that separation rate of Alternaria, Pithomyces chartarum, Fusarium and Aspergillus were higher, particularly, Pithomyces chartarum was significantly identical. Pathological anatomy and histology indicated that the disease likely attacked merino ewes with the age of 6 months old. The clinical manifestations were characterized by inflammational edema in face (ears and eyelids) and mandibular area. Postmortem examination of dead lambs showed enlargement of liver with yellow white patchs of necrotic lesion and tuberous sclerosis and fibrosis on section. Histologic examination of liver showed extravasated blood, severe lesion of liver cells and bile duct, and fatty degeneration. In sheep, fungal toxin induced the secretion of TNF-α, IL-6 and IL-12. These results revealed that Pithomyces chartarum maybe caused facial eczema and inflammation in sheep. The facial eczema was allergic eczema caused by hepatic dysfunction and hepatonecrosis.

Quercetin protects cadmium-induced renal injuries in mice by inhibiting cell pyroptosis.

The toxic heavy metal cadmium (Cd) has a significant impact on kidney health. Documents manifested that non-toxic flavonoid quercetin can reduce Cd-induced kidney damage by reducing oxidative stress and inhibiting apoptosis, while the effect of quercetin on Cd-induced renal cell pyroptosis has not been elucidated. In this study, we established a model of Cd poisoning treated with quercetin both in vitro and in vivo. Results revealed that quercetin effectively reversed the decrease in Cd-induced cell viability. Furthermore, Cd increased blood urea nitrogen while reducing GPX and SOD levels, caused histopathological injuries in kidney with a significantly elevated cell pyroptosis characterized by enhanced levels of proteins representing assembly (NLRP3) and activation (pro IL-1ß, cleaved IL-1ß, and IL-18) of NLRP3 inflammasome as well as pyroptosis executor (pro caspase-1, cleaved caspase-1). However, quercetin administration alleviated kidney injuries above by decreasing cell pyroptosis. Overall, it suggests that kidney cells are susceptible to pyroptotic cell death due to Cd exposure; while quercetin exhibits protective effects through cell pyroptosis inhibition.

The Prevalence of Klebsiella spp. Associated With Bovine Mastitis in China and Its Antimicrobial Resistance Rate: A Meta-Analysis.

Understanding distribution of bovine mastitis pathogen Klebsiella spp. can contribute to the treatment decision and the control within programs of bovine mastitis, we conducted a meta-analysis to investigate the epidemiology and antimicrobial resistance rates of Klebsiella spp. associated with bovine mastitis in China. Three databases, namely, PubMed, Google scholar, and China National Knowledge Infrastructure database, were utilized to obtain relevant publications. According to PRISMA reporting standards, a total of 38 publications were included in the research, among them, 7 papers included an AMR test. The pooled prevalence of Klebsiella spp. was 5.41% (95% CI: 3.87-7.50%). Subgroup analysis revealed that the prevalence was higher in South China (8.55%, 95% CI: 3.57-19.09%) than in North China (4.22%, 95% CI: 2.46-7.14%), in 2010-2020 (7.45%, 95% CI: 5.29-110.40%) than in 2000-2010 (3.14%, 95% CI: 1.90-15.14%), and in the clinical bovine mastitis cases (7.49%, 95% CI: 3.71-14.54%) than in the subclinical cases (4.03%, 95% CI: 1.55-10.08%). The pooled AMR rate revealed that Klebsiella spp. were most resistant to sulfonamides (45.07%, 95% CI: 27.72-63.71%), followed by tetracyclines (36.18%, 95% CI: 23.36-51.34%), aminoglycosides (27.47%, 95% CI: 17.16-40.92%), ß-lactams (27.35%, 95% CI: 16.90-41.05%), amphenicol (26.82%, 95% CI: 14.17-44.87%), lincosamides (21.24%, 95% CI: 7.65-46.75%), macrolides (20.98%, 95% CI: 7.20-47.58%), polypeptides (15.51%, 95% CI: 6.46-32.78%), and quinolones (7.8%, 95% CI: 3.25-17.56%). The climate difference between South and North China and the natural pathogenicity of Klebsiella spp. may be the primary reasons for its distribution, and the prevalence of Klebsiella spp. indicated that the genus is an increasing hazard to the dairy industry. The prevalence of AMR in China is commonly higher than in the European countries and Canada, this is a very important concern for strategy programs to control bovine mastitis caused by Klebsiella spp. in China.

Autophagy Promotes α-Amanitin-Induced Apoptosis of Hepa1-6 Liver Cells.

It is estimated that 90% of deaths from food poisoning in China can be attributed to Amanita poisoning, whose main toxin is α-amanitin. Studies showed that apoptosis plays a critical role in liver injuries induced by α-amanitin. Although the relationship between autophagy and apoptosis in different liver models has been addressed many times, whether autophagy plays a pro or con effect on α-amanitin-induced apoptosis has not been clarified. Therefore, this study was conducted to explore the effect of autophagy in α-amanitin-induced apoptosis in Hepa1-6 liver cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was applied to determine cell viability, a 2',7'-dichlorofluorescin diacetate probe was used to monitor reactive oxygen species (ROS) levels, a flow cytometer and dansylcadaverine (MDC) staining were used to observe α-amanitin-induced apoptosis and autophagy, respectively, and apoptosis and autophagy proteins were assessed by western blotting. The results showed that α-amanitin suppressed cell viability in a time- and concentration-dependent manner. Moreover, the release of ROS was increased with increasing α-amanitin amount. Cell apoptosis and autophagy were noticed and characterized by the increased apoptosis rate and autophagic vesicles under a fluorescence microscope as well as upregulation of Bax/Bcl-2, cleaved caspase-3, and LC3-II/I and downregulation of p62. Further, the autophagy activator rapamycin (Rap) and the inhibitor 3-methylademine (3-MA) were introduced, which showed that the apoptosis rate and the ratio of Bax/Bcl-2 as well as the protein expression level of cleaved caspase-3 increased significantly with the pretreatment of Rap and decreased remarkably with the pretreatment of 3-MA. Moreover, cell viability was found to decrease further with the promotion of autophagy. Notably, the ROS level was attenuated after autophagy was elevated. In conclusion, autophagy could promote α-amanitin-induced Hepa1-6 cell apoptosis, and the process is unassociated with ROS levels. This research provides a theoretical basis for the study of the toxicological mechanism of α-amanitin-induced liver injuries.

Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro.

Mycoplasma species are the smallest prokaryotes capable of self-replication. To investigate Mycoplasma induced autophagy in mammalian cells, Mycoplasma bovis (M. bovis) and bovine mammary epithelial cells (bMEC) were used in an in vitro infection model. Initially, intracellular M. bovis was enclosed within a membrane-like structure in bMEC, as viewed with transmission electron microscopy. In infected bMEC, increased LC3II was verified by Western blotting, RT-PCR and laser confocal microscopy, confirming autophagy at 1, 3 and 6 h post-infection (hpi), with a peak at 6 hpi. However, the M. bovis-induced autophagy flux was subsequently blocked. P62 degradation in infected bMEC was inhibited at 3, 6, 12 and 24 hpi, based on Western blotting and RT-PCR. Beclin1 expression decreased at 12 and 24 hpi. Furthermore, autophagosome maturation was subverted by M. bovis. Autophagosome acidification was inhibited by M. bovis infection, based on detection of mCherry-GFP-LC3 labeled autophagosomes; the decreases in protein levels of Lamp-2a indicate that the lysosomes were impaired by infection. In contrast, activation of autophagy (with rapamycin or HBSS) overcame the M. bovis-induced blockade in phagosome maturation by increasing delivery of M. bovis to the lysosome, with a concurrent decrease in intracellular M. bovis replication. In conclusion, although M. bovis infection induced autophagy in bMEC, the autophagy flux was subsequently impaired by inhibiting autophagosome maturation. Therefore, we conclude that M. bovis subverted autophagy to promote its intracellular replication in bMEC. These findings are the impetus for future studies to further characterize interactions between M. bovis and mammalian host cells.

Facile hydrothermal synthesis of Ti3C2Tx-TiO2 nanocomposites for gaseous volatile organic compounds detection at room temperature.

The gaseous volatile organic compounds (VOCs) sensors with high-selectivity and low-power consumption have been expected for practical applications in environmental monitoring and disease diagnosis. Herein, we demonstrate a room-temperature VOCs gas sensor with enhanced performance based on Ti3C2Tx-TiO2 nanocomposites. The Ti3C2Tx-TiO2 nanocomposites with regular morphology are successfully synthesized via a facile one-step hydrothermal synthesis strategy by using Ti3C2Tx itself as titanium source. Attributed to the formation of interfacial heterojunctions and the modulation of carrier density, the Ti3C2Tx-TiO2 sensor exhibits about 1.5-12.6 times enhanced responses for the detection of various VOCs at room temperature than pure MXene sensor. Moreover, the nanocomposite sensor has better response to hexanal, both an air pollutant and a typical lung cancer biomarker. The gas response of the Ti3C2Tx-TiO2 sensor towards 10 ppm hexanal is about 3.4%. The hexanal gas sensing results display that the nanocomposite sensor maintains a high signal-to-noise ratio and the lower detection limit to hexanal gas is as low as 217 ppb. Due to the low power consumption and easy fabrication process, the Ti3C2Tx-TiO2 nanocomposite sensor is promising for application in IoT environmental monitoring as well as real-time health monitoring.

Mycoplasma bovis-generated reactive oxygen species and induced apoptosis in bovine mammary epithelial cell cultures.

Mycoplasma bovis is an important cause of bovine mastitis in China and worldwide. We hypothesized that M. bovis damages bovine mammary epithelial cells (bMEC), with the degree of damage varying among field isolates. Our objective was to evaluate 2 novel sequence type (ST) field strains of M. bovis (ST172 and ST173) for their ability to induce oxidative stress, cytotoxicity, pathomorphological changes, and apoptosis in bMEC, as a model for pathogenesis of M. bovis-induced bovine mastitis. Cytotoxicity (as indicated by release of lactate dehydrogenase, LDH) from bMEC depended on multiplicity of infection (MOI), with a high MOI (1:1,000) being required to induce cytotoxicity. Morphological changes in bMEC, including shrinkage, loss of cell integrity, and heavy staining (hematoxylin and eosin) of cytoplasm were apparent 24 h after infection with ST172 or ST173 M. bovis, with more severe changes being induced by the latter strain. Adhesion and invasion assays both had curvilinear patterns, peaking 12 h after infection with MOI of 1:1,000. Both production of reactive oxygen species (ROS) and proportion of apoptotic cells increased with time after infection. Increased Bax/Bcl-2 ratios and activation of caspase-3 implied involvement of mitochondria-dependent pathways of apoptosis. Furthermore, intracellular ROS generation, apoptosis, and cleaved caspase-3 were mitigated by N-acetyl-l-cysteine, a ROS scavenger. Both interleukin (IL)-1ß and IL-6 were significantly upregulated by ST172 and ST173 M. bovis, with little change in expression of tumor necrosis factor-α. One ST173 M. bovis isolate had the greatest cytotoxicity of all of our field isolates, with the highest LDH release, adhesion, invasion, ROS production, and apoptosis. In conclusion, our hypothesis was supported: M. bovis damaged bMEC by generating ROS and initiating a mitochondria-dependent pathway of apoptosis, with the degree of damage varying among field isolates. This study provided new knowledge regarding pathogenesis of M. bovis-induced bovine mastitis.