ObjectiveTo investigate the mechanism of action of modified Shaofu Zhuyutang in antagonizing cellular pyroptosis and fibrosis in ectopic endometrial tissues of endometriosis through the Toll-like receptor 4/nuclear factor-κB/NOD-like receptor protein 3 （TRL4/NF-κB/NLPR3） signaling pathway. MethodsSeventy-two SPF-grade female SD rats were randomly divided into a sham-operated group （n = 12） and a modeling group （n = 60）. The rats in the sham-operated group underwent a caesarean section， while the rats in the modeling group were used to establish an endometriosis model through the auto-transplantation method. After successful modeling， the animals were randomly divided into the model group， progesterone group （0.25 mg·kg^-1）， and modified Shaofu Zhuyutang low-， medium-， and high-dose groups （7.5， 15， 30 g·kg^-1）， with 12 animals in each group. After 4 weeks of drug administration， voluntary activity and heat pain latency were observed. The rats were sacrificed for tissue collection， and Masson staining were used to observe histopathological changes in the endometrial tissues. Enzyme-linked immunosorbent assay （ELISA） was used to measure serum levels of interleukin-18 （IL-18）， interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and transforming growth factor-β （TGF-β）. Immunohistochemistry （IHC） was used to detect the protein expression area of tumor necrosis factor-related factor 6 （TRAF6） and NLPR3 in the endometrial tissues. Immunofluorescence （IF） was used to detect the relative fluorescence intensity of Caspase-1 and gasdermin D （GSDMD） in the endometrial tissues. Western blot was employed to measure the relative expression of TRL4， myeloid differentiation factor 88 （MyD88）， TRAF6， NF-κB p65， phosphorylated NF-κB p65 （p-NF-κB p65）， and NLPR3 proteins in endometrial tissues. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 in the endometrial tissues. ResultsCompared with the sham-operated group， rats in the model group showed reduced voluntary activity and shorter heat pain latency. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were elevated. The relative expression areas of TRAF6 and NLPR3 proteins were increased， and the relative fluorescence intensity of Caspase-1 and GSDMD was enhanced. The relative expression of TRL4， MyD88， TRAF6， NF-κB p65， p-NF-κB p65， and NLPR3 proteins， along with the expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA， were significantly increased （P<0.01）. Compared with the model group， rats in the progesterone group and the modified Shaofu Zhuyutang medium- and high-dose groups exhibited improved voluntary activity， longer heat pain latency， the fibrosis of endometrial tissue is alleviated. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were decreased. The relative expression areas of TRAF6 and NLPR3 proteins decreased， and the relative fluorescence intensity of Caspase-1 and GSDMD weakened. The relative expression of TRL4， MyD88， TRAF6， p-NF-κB p65， NLPR3 proteins， and TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA expression were reduced （P<0.05， P<0.01）. ConclusionModified Shaofu Zhuyutang may play a therapeutic role in endometriosis by interfering with key proteins in the TRL4/NF-κB/NLPR3 signaling pathway， reducing NLRP3 inflammasome-induced cellular pyroptosis， antagonizing the fibrosis process in ectopic endometrial tissues， improving the inflammatory microenvironment in the pelvic cavity， and alleviating pain.

ObjectiveTo observe the effects of modified Shaofu Zhuyutang on key proteins of the epidermal growth factor receptor （EGFR）/phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway in SD rats with endometriosis. MethodsAfter successful establishment of an endometriosis model in 60 female SD rats of SPF grade via the auto-transplantation method， the rats were randomly divided into a model group， modified Shaofu Zhuyutang high-， medium-， and low-dose groups， and a gestrinone group， with another 12 rats serving as a blank group. The blank and model groups were administered 10 mL·kg^-1 normal saline， while the high-， medium-， and low-dose groups received 30， 15， and 7.5 g·kg^-1 modified Shaofu Zhuyutang， respectively. The gestrinone group was administered 0.25 mg·kg^-1 gestrinone suspension. After four weeks of treatment， uterine contractions were induced with 2 U of oxytocin， and the writhing response of rats was observed. After 24 h， the rats were euthanized， and the weight and volume of ectopic endometrial tissue were recorded. Hematoxylin-eosin （HE） staining was used to observe pathological changes in endometrial tissues， while the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay was used to evaluate the apoptosis rate of endometrial tissues. Immunofluorescence was used to detect the relative expression areas of the B-cell lymphoma-2 gene-associated promoter （Bad） and B-cell lymphoma-2 （Bcl-2） proteins in endometrial tissues. Serum levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， epidermal growth factor （EGF）， and EGFR were measured by enzyme-linked immunosorbent assay （ELISA）. The relative protein expression levels of EGFR， PI3K， phosphorylated PI3K （p-PI3K）， Akt， and phosphorylated Akt （p-Akt） in endometrial tissues were analyzed by Western blot. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of EGFR， PI3K， and Akt. ResultsCompared with the blank group， the model group showed endometrial thickening， glandular and mesenchymal hyperplasia， a significant decrease in the relative expression area of Bad in ectopic endometrial tissues， a significant increase in the relative expression area of Bcl-2， and a significant reduction in the apoptosis rate as indicated by TUNEL staining. Serum levels of IL-1β， IL-6， TNF-α， EGF， and EGFR were significantly elevated （P<0.01）. The relative protein expression levels of EGFR， PI3K， p-PI3K， Akt， and p-Akt， as well as the mRNA expression levels of EGFR， PI3K， and Akt， were also significantly increased （P<0.01）. Compared with the model group， the high- and medium-dose groups of modified Shaofu Zhuyutang and the gestrinone group exhibited reduced glandular and mesenchymal hyperplasia to varying degrees， with dilated glandular lumens. The number of writhing responses was significantly reduced， the latency to writhing response was significantly prolonged， and the weight and volume of ectopic endometrial tissue were significantly decreased. The relative expression area of Bad in ectopic endometrial tissue was significantly increased， the relative expression area of Bcl-2 was significantly decreased， and the apoptosis rate was significantly elevated as shown by TUNEL staining. Serum levels of IL-1β， IL-6， TNF-α， EGF， and EGFR were significantly reduced， and the relative protein expression levels of EGFR， PI3K， p-PI3K， Akt， and p-Akt， as well as the mRNA expression levels of EGFR， PI3K， and Akt， were significantly decreased （P<0.05，P<0.01）. ConclusionModified Shaofu Zhuyutang may exert therapeutic effects on endometriosis by interfering with key proteins of the EGFR/PI3K/Akt signaling pathway and inducing apoptosis in ectopic endometrial tissue.

ObjectiveTo investigate the mechanism of modified Shaofu Zhuyutang in inducing ferroptosis in ectopic endometrial tissues of rats with endometriosis through the murine double minute 4 （MDM4）/tumor suppressor p53/glutathione peroxidase 4 （GPX4） signaling pathway. MethodsSeventy SPF-grade female SD rats were randomly divided into a blank group （n = 10）， a sham-operated group （n = 10）， and a modeling group （n = 50）. The sham-operated group underwent laparotomy， while the modeling group was subjected to the autotransplantation method to establish an endometriosis model. After successful modeling， the animals were randomly assigned to the model group， progesterone group （0.25 mg·kg^-1）， and modified Shaofu Zhuyutang high-， medium-， and low-dose groups （30， 15， and 7.5 g·kg^-1， respectively）， with 10 rats per group. After four weeks of drug administration， the rats were euthanized for sample collection. The weight and volume of ectopic endometrial tissues were recorded for each group. Transmission electron microscopy （TEM） was employed to observe ultrastructural changes in endometrial tissues， while Prussian blue staining was used to assess iron ion deposition. Serum levels of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. The relative levels of Fe²⁺， malondialdehyde （MDA）， and glutathione （GSH） in endometrial tissues were determined by colorimetric assay. Immunofluorescence （IF） was used to detect the relative fluorescence intensities of GSH and GPX4 in endometrial tissues. The relative expression levels of phosphatidylinositol 3-kinase （PI3K）， phosphorylated PI3K （p-PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， MDM4， p53， and GPX4 proteins were detected by Western blot. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to assess the mRNA expression of PI3K， Akt， MDM4， p53， and GPX4. ResultsCompared with the blank and sham-operated groups， the model group exhibited reduced ferroptotic damage in ultrastructural observations， decreased ferroptotic aggregates and positive iron ion expression area on Prussian blue staining， elevated serum IL-6， IL-1β， and TNF-α levels， reduced Fe²⁺ and MDA content， increased GSH content in endometrial tissues， and enhanced GSH and GPX4 fluorescence intensities （P<0.01）. The protein and mRNA expression levels of PI3K， p-PI3K， Akt， p-Akt， MDM4， and GPX4 were elevated， while those of p53 were decreased （P<0.01）. Compared with the model group， in the progesterone group and the modified Shaofu Zhuyutang high- and medium-dose groups， ferroptotic damage in ultrastructural observations was exacerbated to varying degrees by TEM， and ferroptotic aggregates and positive iron ion expression areas were increased on Prussian blue staining. Serum IL-6， IL-1β， and TNF-α levels decreased， Fe²⁺ and MDA content increased， and GSH content decreased in endometrial tissues. GSH and GPX4 fluorescence intensities weakened， while the protein and mRNA expression levels of PI3K， p-PI3K， Akt， p-Akt， MDM4， and GPX4 decreased， and those of p53 increased （P<0.05， P<0.01）. Conclusionmodified Shaofu Zhuyutang may exert therapeutic effects in endometriosis by inducing ferroptosis in ectopic endometrial tissues， alleviating inflammatory responses， and modulating key proteins in the MDM4/p53/GPX4 signaling pathway.

The Janus kinase/signal transducers and activators of transcription (JAK-STAT) control natural killer (NK) cells development and cytotoxic functions, however, whether long non-coding RNAs (lncRNAs) are involved in this pathway remains unknown. We found that miR155HG was elevated in activated NK cells and promoted their proliferation and effector functions in both NK92 and induced-pluripotent stem cells (iPSCs)-derived NK (iPSC-NK) cells, without reliance on its derived miR-155 and micropeptide P155. Mechanistically, miR155HG bound to miR-6756 and relieved its repression of JAK3 expression, thereby promoting the JAK-STAT pathway and enhancing NK cell proliferation and function. Further investigations disclosed that upon cytokine stimulation, STAT3 directly interacts with miR155HG promoter and induces miR155HG transcription. Collectively, we identify a miR155HG-mediated positive feedback loop of the JAK-STAT signaling. Our study will also provide a power target regarding miR155HG for improving NK cell generation and effector function in the field of NK cell adoptive transfer therapy against cancer, especially iPSC-derived NK cells.

Poly(ADP-ribosyl)ation (PARylation) is a specific form of post-translational modification (PTM) predominantly triggered by the activation of poly-ADP-ribose polymerase 1 (PARP1). However, the role and mechanism of PARylation in the advancement of acute kidney injury (AKI) remain undetermined. Here, we demonstrated the significant upregulation of PARP1 and its associated PARylation in murine models of AKI, consistent with renal biopsy findings in patients with AKI. This elevation in PARP1 expression might be attributed to trimethylation of histone H3 lysine 4 (H3K4me3). Furthermore, a reduction in PARylation levels mitigated renal dysfunction in the AKI mouse models. Mechanistically, liquid chromatography-mass spectrometry indicated that PARylation mainly occurred in receptor for activated C kinase 1 (RACK1), thereby facilitating its subsequent phosphorylation. Moreover, the phosphorylation of RACK1 enhanced its dimerization and accelerated the ubiquitination-mediated hypoxia inducible factor-1α (HIF-1α) degradation, thereby exacerbating kidney injury. Additionally, we identified a PARP1 proteolysis-targeting chimera (PROTAC), A19, as a PARP1 degrader that demonstrated superior protective effects against renal injury compared with PJ34, a previously identified PARP1 inhibitor. Collectively, both genetic and drug-based inhibition of PARylation mitigated kidney injury, indicating that the PARylated RACK1/HIF-1α axis could be a promising therapeutic target for AKI treatment.

Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

Naringenin (4,5,7-trihydroxyflavonoid) is a naturally occurring bioflavonoid found in citrus fruits, which plays an important role in metabolic syndrome, neurological disorders, and cardiovascular diseases. However, the pharmacological mechanism and biological function of naringenin on anti-angiogenesis and anti-tumor immunity have not yet been elucidated. Our study firstly demonstrates that naringenin inhibits the growth of hepatocellular carcinoma (HCC) cells both in vivo and in vitro. Naringenin diminishes the ability of HCC cells to induce tube formation and migration of human umbilical vein endothelial cells (HUVECs) and suppresses neovascularization in chicken chorioallantoic membrane (CAM) assays. Meanwhile, in vivo results demonstrate that naringenin can significantly upregulate level of CD8⁺ T cells, subsequently increasing the level of immune-related cytokines in the tumor immune microenvironment. Mechanistically, we found that naringenin facilitate the K48-linked ubiquitination and subsequent protein degradation of vascular endothelial growth factor A (VEGFA) and mesenchymal-epithelial transition factor (c-Met), which reduces the expression of programmed death ligand 1 (PD-L1). Importantly, combination therapy naringenin with PD-L1 antibody or bevacizumab provided better therapeutic effects in liver cancer. Our study reveals that naringenin can effectively inhibit angiogenesis and anti-tumor immunity in liver cancer by degradation of VEGFA and c-Met in a K48-linked ubiquitination manner. This work enlightens the potential effect of naringenin as a promising therapeutic strategy against anti-angiogenesis and anti-tumor immunity in HCC.

Objective To analyze the characteristics of platelet changes and their influencing factors during postoperative hospitalization in patients who underwent transcatheter aortic valve implantation (TAVI). Methods The patients who underwent TAVI at Beijing Anzhen Hospital Valve Surgery Center between March 2017 and October 2021 were retrospectively selected. The patients were divided into a self-limiting group and a non-self-limiting group according to the characteristics of postoperative platelet decline. In addition, the general preoperative data, preoperative and postoperative ultrasound data, intraoperative data, and the use of anticoagulant drugs during the postoperative stay in the hospital were compared between the two groups. Results A total of 249 patients were enrolled in this study. There were 175 (70.3%) patients in the self-limiting group, including 100 males and 75 females, and there were 74 (29.7%) patients in the non-self-limiting group, including 43 males and 31 females, with no statistical difference between the two groups (P=0.863). The mean age of patients was 73.11±8.88 years in the self-limiting group and 71.54±10.39 years in the non-self-limiting group (P=0.231). The decline of platelets in the self-limiting group generally occurred on the postoperative day 2 and reached the lowest count on the postoperative day 4, and returned to the baseline level on the postoperative day 5-7, while the platelets in the non-self-limiting group changed by simple rise, fall or irregular fluctuation. Patients in the self-limiting group had severer preoperative aortic stenosis (P<0.001) and used more extracorporeal circulation assistance during surgery (P<0.001). Postoperatively, patients in the self-limiting group were more likely to have periaortic valve leakage than those in the non-self-limiting group (P=0.013). Conclusion Platelet changes in most patients after TAVI show a self-limiting decline, which may be related to the severity of patients’ preoperative aortic stenosis, intraoperative extracorporeal circulation device use, and postoperative perivalvular leakage.

Objective: To evaluate the risk of fine particulate matter (PM2.5) on excess mortality among residents. Methods: The data of residential mortality in Yangzhou City, Jiangsu Province from 2015 to 2021 were collected from the Chinese Disease Prevention and Control Information System. The average daily mass concentration of PM2.5 and meteorology data were collected from the Yangzhou Environmental Monitoring Station and Yangzhou Meteorological Bureau, respectively. The effects of PM2.5 on non-accidental mortality, morality of respiratory diseases and mortality of circulatory diseases were evaluated using a generalized additive model. The risk of excess mortality was evaluated using excess risk (ER) and the number of excess mortality. Results: The median average annual mass concentration of PM2.5 was 38.00 (interquartile range, 31.95) µg/m3 in Yangzhou City from 2015 to 2021, decreasing from 51.75 (interquartile range, 32.82) µg/m3 in 2015 to 28.00 (interquartile range, 23.42) µg/m3 in 2021. The median average annual number of non-accidental mortality, mortality of respiratory diseases and mortality of circulatory diseases were 96 (interquartile range, 22), 9 (interquartile range, 5) and 38 (interquartile range, 13) cases, respectively. The greatest effects of per 10 μg/m3 increase in PM2.5 mass concentration on non-accidental mortality, mortality of respiratory diseases, and mortality of circulatory diseases were seen at a cumulative lag of 1 day (ER=0.528%, 95%CI: 0.293%-0.763%), a cumulative lag of 2 days (ER=0.917%, 95%CI: 0.125%-1.714%) and a cumulative lag of 1 day (ER=0.595%, 95%CI: 0.232%-0.961%), respectively. The number of excess mortality caused by PM2.5 on non-accidental mortality, mortality of respiratory diseases, and mortality of circulatory diseases in Yangzhou City from 2015 to 2021 were 2 125, 412 and 977 cases, respectively; compared with 2015, the number of excess mortality in 2021 decreased by 66.95%, 75.53% and 64.42%, respectively. Conclusions An increase in the mass concentration of atmospheric PM2.5 may elevate the risk of excess mortality among residents. Compared to 2015, the number of excess deaths attributed to exposure to atmospheric PM2.5 declined in 2021.

Objective To investigate the potential molecular mechanism of modified Shaofu Zhuyu Decoction in treating endometriosis from the perspective of autophagy in ectopic endometrial tissue cells mediated by the epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Methods Seventy-two female SD rats were divided into the blank group,the model group,the gestrinone group and the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups according to the random number table method,12 rats in each group. Construction of an endometriosis model used auto-transplantation method. The rats in the gestrinone group were gavaged with 0.25mg/(kg·d) of gestrinone suspension,the rats in the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups were gavaged with 30,15,and 7.5g/(kg·d) of modified Shaofu Zhuyu Decoction,respectively,and the rats in the blank and model groups were gavaged with an equal amount of physiological saline,respectively. 4 weeks of continuous treatment. At the end of drug administration,2 U of oxytocin was given to each rat to induce contractions,and the pain of the rats was observed;the weight and volume of ectopic endometrial tissue of the rats were recorded. Hematoxylin-eosin (HE) staining method was used to observe the pathological changes of rat endometrial tissue in each group;transmission electron microscope was used to observe the ultrastructure of rat endometrial tissue. Enzyme-linked immunosorbent assay was used to detect the serum levels of oestradiol (E2),progesterone (P),interleukin-1β (IL-β),interleukin-6 (IL-6),tumour necrosis factor-α (TNF-α),transforming growth factor-β (TGF-β),epidermal growth factor (EGF),and EGFR in the rat serum of each group. Mean fluorescence intensity of autophagy-associated protein yeast Atg6 homologue (Beclin-1) and microtubule-associated protein 1 light chain 3 (LC3B) in endometrial tissues of rat endometrium in each group by immunofluorescence assay. Protein expression levels of EGFR,PI3K,phosphorylated phosphatidylinositol 3-kinase (p-PI3K),AKT,and phosphorylated protein kinase B (p-AKT) in endometrial tissues of rats in each group were examined by protein blotting. Real-time fluorescence PCR was used to detect EGFR,PI3K,and AKT mRNA expression levels.Results Compared with the blank group,the rats in the model group showed typical torsion reaction,ectopic endometrial tissue was visible in the abdominal wall,and the HE result showed thickening of the ectopic endometrial tissue,mesenchymal hyperplasia,and glandular dilatation;only autophagic vesicles were seen in the ultrastructure,and no obvious autophagic structures were seen in some of the fields of view;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,and EGFR levels were elevated and P levels were decreased in rats;autophagy-related proteins Beclin-1 and LC3B decreased in average fluorescence intensity;protein expressions of EGFR,PI3K,AKT,p-PI3K,p-AKT was elevated;and mRNA expressions of EGFR,PI3K,AKT was elevated (P<0.05). Compared with the model group,the gestrinone group and modified Shaofu Zhuyu Decoction high-,medium-dose groups showed a decrease in the number of torsion and a prolongation of the latency time of the torsion response,a decrease in the volume and mass of ectopic endometrial tissues,a decrease in the degree of pathological damage as shown by HE,and the appearance of lysosomes,autophagolysosomes and autophagic vesicles in different degrees in the ultrastructure;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,EGFR levels were decreased,and P levels were increased;the mean fluorescence intensity of autophagy-related proteins Beclin-1 and LC3B was increased;the protein expression of EGFR,PI3K,AKT,p-PI3K,and p-AKT was decreased;and the mRNA expression of EGFR,PI3K,and AKT was decreased (P<0.05). Conclusion The mechanism of modified modified Shaofu Zhuyu Decoction for the treatment of endometriosis may be related to the induction of autophagy of ectopic endometrial tissues mediated by the EGFR/PI3K/AKT signaling pathway and the attenuation of inflammatory responses.