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Journal Pre-proof The Chinese herbal prescription JZ-1 induces autophagy to protect against herpes simplex Virus-2 in human vaginal epithelial cells by inhibiting the PI3K/Akt/mTOR pathway Abstract The Chinese herbal prescription JZ-1 induces autophagy to protect against herpes simplex Virus-2 in human vaginal epithelial cells by inhibiting the PI3K/Akt/mTOR pathway, Journal of Ethnopharmacology (2020), doi: https://doi. Genital herpes is one of the most common, persistent and highly contagious The results were analyzed by the 2 −∆∆CT method with β-actin as an internal control. The sequences of the primers used in this study are listed in Table 2 . anti-p-mTOR, anti-mTOR, anti-gD, anti-ICP5 and anti-β-actin) at 4°C. After washing 267 three times with TBST for 10 min each, the membranes were incubated with 268 secondary antibodies for 1 h at room temperature, washed another three times, and 269 then visualized by an Odyssey Infrared Imaging system (LI-COR Biosciences, USA). The band densities were quantified by ImageJ (National Institutes of Health, USA). All the results are presented as the means ± standard deviation (SD) and were 289 analyzed using SPSS 23.0 software. One-way analysis of variance (ANOVA) and 290 Dunnett's t-test were used to evaluate the significant differences among the groups, 291 and p < 0.05 was considered statistically significant. To determine the appropriate timepoint for the virus infection, we exposed 303 VK2/E6E7 cells to HSV-2 for different duration intervals (6-48 h). The western blot 304 results showed that, compared with that of the control group, the expression of 305 endogenous LC3B-II in the HSV-2-infected cells was increased significantly at each The microtubule-associated protein 1 LC3B is synthesized in the form of the 318 LC3B precursor (pro-LC3B), which is proteolytically processed into the cytosolic 319 LC3B-I isoform. When autophagy is induced, LC3B-I is modified into the Baf+HSV-2-treated group (Fig. 4) , which indicated that JZ-1 induced autophagic flux. The above conclusions were confirmed by an increase in Atg5 in the western blot 382 analysis ( Fig. 4A and C) . Altogether, these data demonstrated that JZ-1 has an 383 anti-HSV-2 effect, which may be related to its induction of autophagic flux. Therefore, Rapa also decreased the release of IFN-α, IFN-β and IL-6, but treatment with 3-MA 413 dramatically increased the secretion levels of these cytokines (Fig. 6A) . The kinase mTOR is a complex of two mTOR components, mTORC1 and 417 mTORC2, which have different protein components (Bhaskar and Hay, 2007) . 418 mTORC1 is a major negative regulator of autophagy, and the PI3K/Akt pathway is a major upstream major modulator of mTORC1 (Schmelzle and Hall, 2000) . To 420 investigate the mechanism by which JZ-1 induces autophagy, we assessed the changes 421 in the classic PI3K/Akt/mTOR pathway in the VK2/E6E7 cells. The results from 422 qRT-PCR assays and western blot analyses showed that, compared with that of the 423 control, the expression of PI3K and phosphorylation levels of Akt and mTOR (p-Akt 424 and p-mTOR) in the HSV-2-infected cells were significantly increased, and these 425 proteins all were decreased significantly in the JZ-1+HSV-2 group (Fig. 5B, Fig. 7C ). showed that, compared with that of the HSV-2 group, the expression levels of PI3K, 433 p-Akt and p-mTOR were significantly reduced in the LY + HSV-2 group, indicating 434 that it indeed inhibited the PI3K/Akt/ mTOR pathway (Fig. 6C, Fig. 7D ). On this 435 basis, we measured the expression levels of LC3B and Atg5 and found that, in the 436 JZ-1 +HSV-2 +LY -treated group, the LC3B and Atg5 mRNA expression levels and 437 the LC3B-II and Atg5 protein expression levels were not different from those in the 438 LY+HSV-2 group (Fig. 6C, Fig. 7D ). As expected, LY pretreatment eliminated the The present study demonstrated that JZ-1 is effective in combating HSV-2 611 infection, and the potential mechanism is associated with inducing autophagy through 612 inhibiting the PI3K/Akt/mTOR pathway. Our findings suggest that JZ-1 is a 613 promising anti-HSV-2 formula to be further researched and developed. Table 2 The