mtROS-mediated Akt/AMPK/mTOR pathway was involved in Copper-induced autophagy and it attenuates Copper-induced apoptosis in RAW264.7 mouse monocytes.

Copper (Cu) is a trace element necessary in animals as well as human beings. However, excessive Cu is toxic to immunocytes, but the precise mechanism is largely unclear so far. This work was conducted aiming to examine the Cu-mediated autophagy mechanism together with its role in Cu toxicology in RAW264.7 cells. Here, we demonstrated that CuSO4 reduced the cell viability depending on its dose. CuSO4 could obviously increase autophagy in RAW264.7 cells. According to the obtained results, CuSO4 induced autophagy through Akt/AMPK/mTOR pathway which characterized by down regulation of p-Akt (Ser473)/Akt, p-mTOR/mTOR, p-ULK1(Ser757)/ULK1 and subsequent up-regulation of p-AMPKα/AMPKα and p-ULK1(Ser555)/ULK1. Furthermore, CuSO4 significantly induced the production of mitochondrial reactive oxygen species (mtROS). In addition, CuSO4-mediated apoptosis and autophagy might be suppressed through suppressing mtROS generation by exposure to Mito-TEMPO. Intriguingly, autophagy promotion with rapamycin could decrease the apoptosis and the inhibition of autophagy with knock down Atg5 could enhance the apoptosis induced by CuSO4. Moreover, our results suggested that mtROS is the original cause in CuSO4-induced apoptosis and autophagy. Additionally, CuSO4 induced autophagy through mtROS-dependent Akt/AMPK/mTOR signalling pathwayin RAW264.7 cells. Moreover, autophagy activation might potentially generate a protection mechanism for improving CuSO4-induced RAW264.7 cell apoptosis.

Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆TK/∆gE.

The outbreak of pseudorabies in China, caused by more virulent pseudorabies virus (PRV) than the classical strains, has led to considerable economic losses. In this study, PRV strain HNXY was isolated from the Henan province of China in 2015 from the pig farm with severe reproductive failure in sows and a high mortality in piglets. The 50% tissue culture infectious doses (TCID50) of HNXY in Vero cells were examined to be 106.5/mL, and the neutralisation titer against Bartha-K61 was significantly higher than against HNXY when tested with the serum from Bartha-K61 vaccinated pigs. The 50% lethal doses (LD50) of HNXY to six-week-old BALB/c mice and two-month-old PRV-free pigs were both 102.3 TCID50. HNXY was classified as genotype II, and numerous amino acid variations were found in gB, gE, gC, gD, TK, and RR1 proteins, compared with PRV from other countries or those prevalent in China before 2012. The attenuated rHNXY-∆TK/∆gE was further constructed, which presented significantly smaller plaques than HNXY, as well as the similar growth kinetics. rHNXY-∆TK/∆gE was confirmed to be non-pathogenic to six-week-old BALB/c mice and zero-day-old piglets. This study isolated updated PRV promising to develop into a new vaccine candidate.