To investigate the therapeutic efficacy of artesunate (AS) on polycystic ovary syndrome (PCOS) in mice and explore the potential mechanism primarily.

Twenty-five female C57BL/6J mice were randomly divided into Control group, model group (PCOS group), low- and high-dose AS groups (AS15 and AS30 groups) and metformin group (Met group). In addition to the Control group, the mouse model of PCOS was established by subcutaneous injection of dehydroepiandrosterone (DHEA, 60 mg/kg) following by a high-fat diet for 21 d. After modeling, AS of 15 and 30 mg/kg was intraperitoneally injected into the mice of the AS15 and AS30 groups, respectively, and 200 mg/kg Met was given to those of the Met group by gavage, once per day, for 6 weeks. ELISA was used to detect serum testosterone (T), fasting insulin (FINS), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the LH/FSH ratio was calculated. The levels of fasting blood glucose (FBG), triglyceride (TG) and total cholesterol (TC) were detected by automatic biochemical analyzer, and the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. The estrous cycle was observed, and HE staining was performed for pathological changes in the ovary and uterus. Immunofluorescence assay was employed to measure the expression of p-eIF2α, ATF4 and CHOP in the ovarian tissue. After steroidogenic human granulosa-like tumor cell line KGN were exposed to 100 μmol/L DHEA to simulate the hyperandrogen environment of PCOS, and then treated with 5 and 10 μg/mL AS for 24 h, the protein levels of endoplasmic reticulum stress signaling pathway was detected by Western blotting.

Compared with the Control group, the PCOS mice had disturbed estrous cycle, polycystic changes in the ovaries, and significantly increased serum T level and LH/FSH ratio (P<0.05), and obviously elevated HOMA-IR, TC and TG levels in terms of metabolism (P<0.01). The expression levels of p-eIF2α, ATF4 and CHOP were notably up-regulated in the ovarian granulosa cells of PCOS mice and KGN cells after DHEA exposure (P<0.05). Additionally, AS treatment attenuated the pathological changes of ovary and uterine expression, decreased the serum T level and the LH/FSH ratio (P<0.05), and reduced HOMA-IR, TC and TG levels (P<0.05) when compared with the PCOS mice. Moreover, the expression levels of p-eIF2α, ATF4 and CHOP were significantly down-regulated after AS treatment in both ovarian granulosa cells of PCOS mice and KGN cells (P<0.05).

AS significantly improves glycolipid metabolic disorder and reproductive dysfunction in PCOS mice, which may be associated with its suppressing endoplasmic reticulum stress by inhibiting the PERK/eIF2α/ATF4/CHOP pathway.

To investigate the role of transcription factor EB (TFEB) in endotoxin-tolerant macrophages.

The RAW264.7 cells were divided into blank group (DMEM medium), LPS 5 group (5 ng/mL LPS treatment for 4 h), LPS 100 group (100 ng/mL LPS treatment for 4 h), and tolerance group (5 ng/mL LPS for 12 h followed by 100 ng/mL LPS for 4 h). The releases of inflammatory factors TNF-α and IL-6 were measured using ELISA. Western blotting and immunofluorescence assay were used to evaluate the distribution of autophagy-related proteins LC3 and P62, as well as TFEB in the cytoplasm and nucleus. Lentiviral overexpression of TFEB or siRNA-mediated knockdown of TFEB were performed to observe the changes in autophagy levels and bacterial clearance ability in the tolerant cells.

The cells in the tolerance group had significantly lower contents of TNF-α and IL-6, as well as reduced bacterial clearance ability (P < 0.01), down-regulated LC3 expression while up-regulated P62 level, and decreased expression of TFEB in both the cytoplasm and nucleus (P < 0.01) when compared with the cells of the LPS 100 group. Overexpression of TFEB significantly increased LC3 level, reduced P62 level, and enhanced bacterial clearance ability in the endotoxin-tolerant cells (P < 0.01). In contrast, siRNA-mediated knockdown of TFEB had no significant impacts on LC3 and P62 expression levels or bacterial clearance ability.

Overexpression of TFEB can restore the autophagy of endotoxin-tolerant cells and enhance their bacterial clearance capacity, thereby alleviating the immunosuppressive state of sepsis. These findings suggest that TFEB holds promise as a potential therapeutic target for the prevention and treatment of sepsis.

To investigate the role and underlying mechanism of activating transcription factor 3 (ATF3) in suppressing lipopolysaccharide (LPS)-induced autophagy and inflammatory responses in macrophages.

Firstly, the gene expression omnibus (GEO) database was used to analyze ATF3 expression in peripheral blood mononuclear cells (PBMCs) from sepsis patients, and gene set enrichment analysis (GSEA) was performed to identify enriched signaling pathways. Secondly, RAW264.7 macrophages were divided into a blank control group and an LPS-stimulated group (100 ng/mL LPS). Western blotting and immunofluorescence assay were used to detect ATF3 protein expression and observe its subcellular localization, respectively. Lentiviral transduction was used to generate ATF3 knockdown and overexpression cell lines to evaluate their effects on cytokine release and bacterial clearance. Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing was employed to identify downstream target genes transcriptionally regulated by ATF3. Furthermore, the impact of ATF3 knockdown or overexpression on autophagy-related gene 5 (ATG5), autophagy-related gene 16-like 1 (ATG16L1), and autophagy levels was evaluated.

GEO analysis revealed that ATF3 expression was significantly elevated in PBMCs from sepsis patients (P<0.01), and GSEA showed significant enrichment of autophagy-related and inflammation-related pathways (P<0.01). In RAW264.7 cells, 100 ng/mL LPS stimulation significantly increased ATF3 expression in the nucleus than the blank control group (P<0.01). ATF3 knockdown led to increased secretions of TNF-α and IL-6 and enhanced bacterial clearance of macrophages (P<0.01), whereas ATF3 overexpression significantly suppressed TNF-α and IL-6 releases, and remained bacterial clearance at a low level when compared with the conditions in the negative control (NC) group (P<0.01). CUT&Tag results demonstrated that ATF3 was enriched at the promoter regions of key autophagy genes Atg5 and Atg16l1. Compared with the NC group, ATF3 knockdown significantly up-regulated the protein levels of LC3-Ⅱ/Ⅰ, ATG5, and ATG16L1 while decreased p62 expression (P<0.01). Conversely, ATF3 overexpression inhibited the expression of LC3-Ⅱ/Ⅰ, ATG5, and ATG16L1(P<0.01), but had no significant effect on p62 level.

Sepsis induces elevated ATF3 expression in macrophages, and suppresses autophagic activity and down-regulates proinflammatory cytokines TNF-α and IL-6, which probably mediated by ATF3 regulating transcription of ATG5 and ATG16L1, suggesting ATF3 as a potential therapeutic target for autophagy-inflammation imbalance.

To investigate the mechanism of celastrol（Cel）against estrogen receptor positive（ER⁺）breast cancer by activating endoplasmic reticulum stress-mediated apoptosis.

ER⁺ human breast cancer cell lines MCF-7 and T47D were used as research objects. MTT assay was used to detect the effect of Cel on cell viability. Western blotting were employed to detect the expression of apoptosis-related proteins. RT-qPCR and Western blotting were applied to measure the expression of endoplasmic reticulum stress-related molecules at mRNA and protein levels. A nude mouse model of orthotopic transplantation of MCF-7 cells was established to observe the effect of Cel on the growth of transplanted tumors.

MTT results showed that Cel inhibited the viability of MCF-7 and T47D cells in a dose-dependent manner（P<0.05）. Western blotting showed that Cel up-regulated the expression of apoptosis-related proteins, Cleaved PARP and BAX, and down-regulated the expression of BCL-2（P<0.05）. RT-qPCR indicated that Cel up-regulated the mRNA levels of endoplasmic reticulum stress-related genes IRE1α, ATF6, PERK, ATF4, CHOP and BIP（P<0.05）. Western blotting revealed that Cel treatment resulted in increased expression levels of endoplasmic reticulum stress marker BIP, of PERK pathway related proteins p-PERK, p-eIF2α, ATF4 and CHOP, of IRE1α pathway associated proteins p-IRE1α and XBP1s, and of ATF6 pathway associated protein Cleared ATF6. In vivo experiment results showed that Cel significantly reduced the volume of orthotopic transplanted tumor in nude mice（P<0.05）, and had no significant effect on their body weight.

Cel has a certain inhibitory effect on the proliferation of ER⁺ breast cancer MCF-7 and T47D cells, and its mechanism may be related to the activation of endoplasmic reticulum stress signaling pathway to induce apoptosis.