Acta Biochimica et Biophysica Sinica

Pregnancy is a very complex and delicate process, inflammation in early pregnancy may result in pregnancy loss or defective implantation. Melatonin, mainly produced from the pineal body, which exerts several pharmacological effects. N6-methyladenosine (m6A) is the most prevalent modification of eukaryotic mRNA. The aim of this study was to investigate the association between melatonin and m6A during pregnancy and elaborate the underlying protective mechanism of melatonin during pregnancy. In vitro, melatonin was found to alleviated LPS-induced reductions in the number of implantation sites. Besides, melatonin was found to alleviate the activation of inflammation, autophagy and apoptosis pathways. In vitro studies demonstrated that melatonin regulated several downstream pathways in an m6A-dependent manner via melatonin receptor MTNR1B. Our findings revealed the important roles of m6A in the establishment of pregnancy and discovered a new mechanism of how melatonin protects pregnancy.

The treatment of uterine fibroids and the development of new drugs depend on adeeper understanding of the developmental mechanisms of uterine fibroids. Here, the role of ESR1 and miR-17 on the uterine fibroids cell proliferation and apoptosis and their relationship were investigated in USMCs. Our results showed that ESR1 increased the proliferation of USMCs and inhibited their apoptosis. In addition, ESR1 could directly bind the promoter regionof TP53 and inhibit its expression. MiR-17 increased the apoptosis of USMCs and inhibited their proliferation via decreasing the level of ESR1 by targeting its 3UTR. Our research provides a new understanding of the development of uterine fibroids and provides a theoretical basis for the treatment of uterine fibroids.

SPINK1 has been regarded as a reversible trypsinogen inhibitor for the inappropriate activation of trypsin, a key step in the initiation of acute pancreatitis (AP). However, the mechanisms of its action remains largely unclear and controversial. Here, we reported an unexpected effects of SPINK1 on inhibiting trypsinogen activation through the regulation of impaired autophagy in cerulein-stimulated AR42J cells, a well-established in vitro model of acute pancreatitis. Firstly, we found that the impaired autophagic flux was induced and trypsinogen activity enhanced in the above setting. Then, we showed that SPINK1 overexpression could inhibit the level of increased autophagic activity, improving the hindered autophagy flux, and significantly decreased the trypsinogen activity, whereas shRNA-caused downregulation of SPINK1 exacerbated the impairment of autophagic flux and trypsin activity, in the same cerulein-processed cells. More importantly, the trypsinogen activation in this model could be ameliorated by 3-Methyladenine(3-MA), an autophagy inhibitor. Thus, this study revealed, possibly for the first time, that SPINK1 greatly blocked the trypsinogen activation possibly through the modulation of impaired autophagy in cerulein-induced in vitro model of acute pancreatitis.

Antitumor transcription activator NFE2L1, with the functions to regulate redox homeostasis, protein turnover, and material metabolism, plays an important role in embryonic development and specialization of tissue and organ functions. Deficiency of NFE2L1 gene in different regions yields distinct phenotypes, suggesting that NFE2L1 may have a transcription factor-independent function. Here we originally discovered the non-transcription factor activity of NFE2L1 by constructing a truncated protein-NFE2L1{Delta}C without 152 aa at the C-terminus which lost the transcription factor activity. The regulation of NFE2L1 on redox homeostasis, proteasome function, and immune response mainly depends on its transcription activator function in nucleus, while the regulation on metabolism, ribosome function, and canceration is germanely to its non-transcription factor activity in cytoplasm. Surprisingly, the results indicated the tumor suppressive effect of NFE2L1 by repression of Wnt/{beta}-catenin signaling in a non-transcription factor manner, indicating the potential value of NFE2L1 as a therapeutic target in clinical cancer treatment independent of its transcription factor activity. Our observations reveal the non-transcription factor activity of NFE2L1 for the first time, and lay foundation for the basic and applied research of NFE2L1.

Emodin has a variety of pharmacological functions including anti-bacterial infection, anti-inflammatory, anti-oxidation, anti-tumor, regulation of gastrointestinal activities and anti-hepatic and lung fibrosis. However, the role of emodin in the regulation of renal interstitial fibrosis(RIF) remains poorly understood. In this study, we investigated the regulation of emodin in RIF and revealed the underlying molecular mechanisms. We established a unilateral ureter obstruction(UUO) model to simulate renal interstitial fibrosis in rats. We found that UUO rats were observed a large amount of inflammatory cell infiltration, more fibroblast proliferation, collagen fiber proliferation. Furthermore, we demonstrated that the up-regulation of TIMP1 and down-regulation of MMP9 were related to renal fibrosis. However, those phenomena in emodin-treated UUO rats were ameliorated. Collectively, our results provide new insights into the treatment of RIF and suggest that the TIMP1/MM9 signaling axis may be a potential therapeutic target for RIF.

Pneumonia induced by severe acute respiratory coronavirus 2 (SARS-CoV-2) via ACE2 receptor may affect many organ systems like lung, heart and kidney. An autopsy report revealed positive SARS-Cov-2 detection results in ovary, however, the developmental-stage-specific and cell-type-specific risk in fetal primordial germ cells (PGCs) and adult women ovary remained unclear. In this study, we used single-cell RNA-sequencing (scRNA-seq) datasets spanning several developmental stages of ovary including PGCs and cumulus-oocyte complex (COC) to investigate the potential risk of SARS-CoV-2 infection. We found that PGCs and COC exhibited high ACE2 expression. More importantly, the ratio of ACE2-positive cells was sharply up-regulated in primary stage and ACE2 was expressed in all oocytes and cumulus cells in preovulatory stage, suggesting the possible risk of SARS-CoV-2 infection in follicular development. CatB/L, not TMPRSS2, was identified to prime for SARS-CoV-2 entry in follicle. Our findings provided insights into the potential risk of SARS-CoV-2 infection during folliculogenesis in adulthood and the possible risk in fetal PGCs.

Cardiac hypertrophy is a complex process induced by the activation of multiple signaling pathways. We previously reported that anacardic acid (AA), a histone acetylase (HAT) inhibitor, attenuates phenylephrine (PE)-induced cardiac hypertrophy by downregulating histone H3 acetylation at lysine 9 (H3K9ac). Unfortunately, the upstream signaling events remained unknown. The mitogen-activated protein kinase (MAPK) pathway is an important regulator of cardiac hypertrophy. In this study, we explored the role of JNK/MAPK signaling in cardiac hypertrophy. A mouse model of cardiomyocyte hypertrophy was successfully established in vitro using PE. This study showed that p-JNK directly interacts with HATs (P300 and P300/CBP-associated factor, PCAF) and alters H3K9ac. In addition, both the JNK inhibitor SP600125 and the HAT inhibitor AA attenuated p-JNK overexpression and H3K9 hyperacetylation by inhibiting P300 and PCAF during PE-induced cardiomyocyte hypertrophy. Moreover, we demonstrated that both SP600125 and AA attenuate the overexpression of cardiac hypertrophy-related genes (MEF2A, ANP, BNP, and {beta}-MHC), preventing cardiomyocyte hypertrophy and dysfunction. These results revealed a novel mechanism through which AA might protect mice from PE-induced cardiac hypertrophy. In particular, AA inhibits the effects of JNK signaling on HAT-mediated histone acetylation, and could therefore be used to prevent and treat hypertrophic cardiomyopathy.

Erectile dysfunction (ED) is a male sexual dysfunction with a gradually increasing prevalence, and current treatments are ineffective. This study aims to find a safer and more effective treatment for erectile dysfunction. Bone marrow-derived mesenchymal stem cells (BM-MSCs) treated with VEGFA. The expression of endothelial markers vWF, VE-cadherin and eNOS were determined by overexpressing MALAT1 and CDC42, respectively. The results showed that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs and the expression of endothelial marker-related proteins. Moreover, MALAT1 induced differentiation of BM-MCs to ECs through the CDC42/PAK1/Paxillin pathway was explored by transfecting si-MALAT1 and overexpressing CDC42 as well as PAK1 in the BM-MSCs. Next, miR-206 was overexpressed within BM-MCs to determine CDC42 expression. The binding sites of MALAT1, miR-206 and CDC42 were reported using luciferase. it was found that the MALAT1 competes with CDC42 3-UTR for binding miR-206, which in turn is involved in differentiating BM-MSCs to ECs. Finally, DMED rat modeling success was demonstrated by APO experiments. MALAT1 overexpression-modified BM-MSCs had significant therapeutic effects in DMED rats.

Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1 accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1 target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. In addition, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1 destabilization under normoxic conditions. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1-mediated hypoxic gene activation.

Pancreatic cancer is one of the most aggressive and lethal malignancies with extremely poor prognosis, and KLK7 was considered as a potential therapeutic target. In this study, we analyzed the expression of KLK7 in TCGA and GTEx databases and found that KLK7 had a negative correlation to long-term survival rate (>1.5 years) of pancreatic cancer patients. Compound 42 is a coumarinic derivative, a suicide substrate inhibitor of KLK7, which has been proved to inhibit the proliferation of PANC-1 cells in vitro effectively in our previous study. In this study, we further investigated the inhibition ability of Compound 42 in tumor formation and development in CDX and PDX tumor models of pancreatic cancer subsequently. Besides, we studied the inhibitory mechanism of Compound 42 and the result showed that Compound 42 arrested the pancreatic cancer cell cycle in G0/G1 phase and induced ferroptosis through down-regulation of GPX4 protein level and accumulation of iron ion. Thus, these experiments demonstrate that Compound 42, suppressing pancreatic cancer in vivo, is expected to become a novel drug for pancreatic cancer treatment.

Endometrial natural killer cells (eNK) at the proliferative phase are one of the major lymphocytes in the endometrium, but their phenotype and function remain elucidated. Here, we demonstrated that eNK cells had three subtypes and were different from peripheral blood NK (pbNK) and decidual NK (dNK) cells. They played an important role in maintaining the homeostasis of the endometrium through preventing endometrial stromal cells (ESCs) from the differentiation into myofibroblasts physiologically. However, in the fibrotic endometria of patients with intrauterine adhesions (IUA), eNK cells were significantly decreased and highly negatively correlated with an increase in myofibroblasts. In vitro experiments showed that eNK cells could inhibit the differentiation of ESCs into myofibroblasts and promote the dedifferentiation of myofibroblasts, in which the main effector molecules from eNK cells was Prostaglandin D2 (PGD2). PGD2 downregulated the expression of ZNF521 to decrease profibrotic protein synthesis. Furthermore, we confirmed the anti-endometrial fibrosis effect of eNK cells and its mechanism in an IUA-like murine model. These findings reveal an important role of eNK cells in endometrial homeostasis and provide potential therapeutic approaches for IUA patients.

Mammal cardiomyocytes lose their ability of regeneration shortly after birth. Reduced cardiomyocytes number caused by myocardial damage is unable to reverse in current clinical therapies. Therefore, it is important and urgent to find new approaches to stimulate cardiomyocytes regeneration. Here we design a recombinant protein IGF1-24 and show that it triggers cardiomyocytes proliferation in rat. 7 days after tail intravenous injection of IGF1-24, 6-7-weeks-old healthy rats showed marked improvements in cardiomyocytes proliferation. Next, we injured rats cardiac with isoproterenol and treated them with IGF1-24 injection. We found that it efficiently induced cell proliferation with significant improvements in heart histology. These results show that the recombinant IGF1-24 stimulates cardiomyocytes proliferation and can be used to achieve cardiac repair through stimulating endogenous cardiomyocyte proliferation in rats. The IGF 1-24 could be a prospective medicine to heart repair because it has high efficiency in triggering cell proliferation and it can be easily applied to heart by intravenous injection

Coronavirus disease 2019 (COVID-19) has caused the public health crisis in the whole world. Anti-androgens block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and protect against severe clinical COVID-19 outcomes. GT0918, a novel androgen receptor antagonist, accelerated viral clearance and increased recovery rate in outpatients by blocking SARS-CoV-2 infection though down-regulating ACE2 and TMPRSS2 expression. Further clinical study showed that GT0918 reduced mortality rate and shortened hospital stay in hospitalized COVID-19 patients. GT0918 also exhibits protective efficacy in severe COVID-19 patient in critical care. However, the mechanism of GT0918 treatment for severe COVID-19 disease is unknown. Here, we found GT0918 decreased the expression and secretion of proinflammatory cytokines through NF-{kappa}B signaling pathway. The acute lung injury induced by LPS or Poly(I:C) was also attenuated in GT0918-treated mice, compared with vehicle control group. Moreover, GT0918 elevated the NRF2 protein level but not mRNA transcription activity. GT0918 induced proinflammatory cytokines downregulation was partially dependent on NRF2. In conclusion, our data demonstrate that GT0918 reduced cytokine release and suppressed inflammatory responses through inhibiting NF-{kappa}B signaling and activating NRF2. GT0918 is not only effective for treatment of mild to moderate COVID-19 patients, but also a potential therapeutic drug for severe COVID-19 patients by reducing the risk of cytokine storm and acute respiratory distress syndrome.

The process of epithelial-mesenchymal transition (EMT) is required for the progression of renal interstitial fibrosis (RIF). Ubiquitin-specific protease 4 (USP4) can facilitate development of transforming growth factor, beta 1 (TGF-{beta}1) induced EMT in some cancer cells. However, the role of USP4 in EMT during RIF remains unknown. We aimed to explore the effect of USP4 on the EMT induced by TGF-{beta}1 of renal tubular epithelial cells and involved mechanism in RIF. In vivo, on the 7th and 14th day after unilateral ureteral obstruction (UUO), the expression of USP4 protein in the obstructed kidneys was detected by immunohistochemistry and Western blot assay. In vitro, NRK-52E cells were stimulated with TGF-{beta}1 10ng/ml. The protein expressions of USP4, E-cadherin and alpha smooth muscle actin (-SMA) were detected at different time points by Western blot. After transfected with USP4 siRNA, the cells were cultured with TGF-{beta}1 for additional 24 hours. The expressions of E-cadherin, -SMA, and TGF{beta} receptor type I (T{beta}RI) were detected by immunofluorescence. And the protein expressions of USP4, E-cadherin, -SMA and T{beta}RI were detected by Western blot assay. Compared with sham operation group, the expression of USP4 in UUO model group increased significantly with the prolongation of obstruction time. After NRK-52E was stimulated by TGF-{beta}1, the expression of USP4 protein increased gradually. At 6h, 12h, and 24h, the difference between the experimental group and the control group was statistically significant. At the same time, E-cadherin decreased significantly, while -SMA increased significantly. Compared with the TGF-{beta}1 group, the cells in USP4 siRNA transfection group restored E-cadherin and weakened -SMA expression. At the same time, protein expressions of USP4 and T{beta}RI were also significantly decreased. These data imply that USP4 is a harmful molecule induced by TGF-{beta}1, which plays an important role by upregulating the expression of T{beta}RI and promoting EMT of renal tubular epithelial cells, thereby facilitating renal interstitial fibrosis.

Vascular calcification/aging is a crucial feature of diabetic macro vasculopathy, resulting in serious cardiovascular diseases. The calcification/senescence of vascular smooth muscle cells (VSMCs) induced by hyperglycemia can cause diabetic vascular calcification/aging. However, the mechanism of VSMCs calcification/senescence involved in diabetic vascular calcification/aging remains unknown. The purpose of this study was to determine how the high glucose (HG) information in circulating blood is transmitted from vascular endothelial cells (ECs) to VSMCs, which are not contacted with blood directly. Exosomes have attracted much attention for their vital roles in regulating cell-to-cell communication. In this study, we found that milk fat globule epidermal growth factor 8 (MFGE8) was enriched in high glucose induced human umbilical vein endothelial cell exosomes (HG-HUVEC-Exo) and regulate VSMCs calcification/senescence, characterized by up-regulated expressions of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2), as well as the increased mineralized nodules and senescence-associated {beta}-galactosidase (SA-{beta}-gal) positive cells. Upstream mechanism studies showed that sirtuin1 (SIRT1) was involved in VSMCs calcification/senescence by affecting the expression of MFGE8. We also found that inflammatory response mediated by IL-1{beta}, IL-6, and IL-8 was closely associated with MFGE8 and played a key role in regulating HG-HUVEC-Exo-induced VSMCs calcification/senescence. These findings provide a new insight into the mechanism of exosomal MFGE8 as a potential preventive and therapeutic target for the intervention of diabetic vascular calcification/aging.

Nobiletin is a natural compound useful for the prevention and treatment of several diseases. However, the precise role of nobiletin in heart failure is unclear. Nobiletin treatment prevents pressure overload- and myocardial infarction-induced heart failure. Using affinity purification of biotinylated nobiletin from rat heart cell lysates, we identified sirtuin 5 (SIRT5) as a novel nobiletin-binding protein. Nobiletin enhanced the desuccinylase activity of SIRT5 in vitro. Compared to wild-type mice, SIRT5-overexpressing transgenic mice resisted pressure overload-induced systolic dysfunction. Conversely, SIRT5 knockout disrupted the nobiletin-mediated therapeutic effects on heart failure in mice. SIRT5 desuccinylated p300 at lysine 1568 and reduced the histone acetyltransferase (HAT) activity of p300. The desuccinylated p300 mutant suppressed the phenylephrine-induced cardiomyocyte hypertrophic responses. These findings suggest that nobiletin prevents heart failure development through SIRT5-dependent inhibition of p300-HAT activity. Nobiletin, a nontoxic dietary compound, is a potential therapeutic agent for heart failure in humans.

Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at G1 and G2/M phases, which is thought to be one of the reasons for the potent antitumor activity of splicing inhibitors. However, the molecular mechanisms underlying the cell cycle arrest have many unknown aspects. In particular, the mechanism of G2/M-phase arrest caused by splicing inhibition is completely unknown. Here, we found that lower and higher concentrations of pladienolide B caused M-phase and G2-phase arrest, respectively. We analyzed protein levels of cell cycle regulators and found that a truncated form of the p27 CDK inhibitor, named p27*, accumulates in G2-arrested cells. Overexpression of p27* caused partial G2-phase arrest. Conversely, knockdown of p27* accelerated exit from G2/M phase after washout of splicing inhibitor. These results suggest that p27* contributes to G2/M-phase arrest caused by splicing inhibition. We also found that p27* bound to and inhibited M-phase cyclins, although it is well known that p27 regulates G1/S transition. Intriguingly, p27*, but not full-length p27, was resistant to proteasomal degradation and remained in G2/M phase. These results suggest that p27*, which is a very stable truncated protein in G2/M phase, contributes to G2-phase arrest caused by splicing inhibition.

Setd7, a catalytic enzyme responsible for histone H3K4 methylation, is implicated in various cardiac diseases. However, the role of Setd7 in pathological cardiac hypertrophy remains unclear. In this study, we observed that Setd7 is significantly elevated in pathological hypertrophy stimuli cardiomyocytes and mouse failing hearts. Subsequently, we found that mice lacking Setd7 remarkably preserved cardiac function after transverse aortic constriction, as demonstrated by improving myocardial hypertrophy and fibrosis, whereas Setd7 overexpression in cardiomyocytes deteriorated hypertrophy phenotype. Further in vitro analyses revealed that Setd7 mediated-E2F1 activation induces E3 ubiquitin protein ligases WWP2 expression to catalyze the lipid-peroxide-reducing enzyme GPx4 ubiquitination degradation, ultimately causing widespread lipid peroxidation and boosting pathological cardiac hypertrophy. Remarkably, loss of activity of GPx4 blunted the Setd7 knockdown exerts antihypertrophic effect in pathological cardiomyocytes hypertrophy, further confirming an important role of lipid peroxidation in Setd7-mediated failing hearts. In summary, the role of Setd7 in pressure overload-induced cardiac hypertrophy is regulated by the Setd7-E2F1-WWP2-GPx4 signaling pathway, suggesting that targeting Setd7 is a promising therapeutic strategy to attenuate pathological cardiac hypertrophy and heart failure.

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ER)-positive breast cancer (BCa) therapy. Aberrant alteration in modulation of E2/ER signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Thus, identification the efficient ER cofactor remains necessary for finding a potential therapeutic target for endocrine resistance. Herein, we have demonstrated that Myb like, SWIRM and MPN domains 1 (MYSM1) as a histone deubiquitinase is a novel ER co-activator with established Drosophila experimental model. Our results showed that MYSM1 participated in up-regulation of ER action via histone and non-histone deubiquitination. We provided the evidence to show that MYSM1 was involved in maintenance of ER stability via ER deubiquitination. Furthermore, silencing MYSM1 induced enhancement of histone H2A ubiquitination as well as reduction of histone H3K4me3 and H3Ac levels at cis regulatory elements on promoter of ER-regulated gene. In addition, MYSM1 depletion attenuated cell proliferation/growth in BCa-derived cell lines and xenograft models. Knockdown of MYSM1 increased the sensitivity of antiestrogen agents in BCa cells. MYSM1 was highly expressed in clinical BCa samples, especially in aromatase inhibitor (AI) non-responsive tissues. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ER action and provide a potential therapeutic target for endocrine resistance in BCa.

1.Viral protease is an attractive target for antiviral therapeutics, but current viral protease inhibitor screening methods still need to be improved. Here, we systematically investigated the sites that may accommodate exogenous short peptides within Enhanced Green Fluorescent Protein (EGFP) and constructed a series of recombinant green fluorescent proteins (rGFPs). Meanwhile, a cell-based, simple and reliable assay system named DIFF-rGFP was developed relying on the co-expression of rGFP and the protease for protease inhibitor screening with the example of 3CLpro, in which the fluorescence intensity increases with the action of the inhibitor. The DIFF-rGFP assay avoided the requirement of a higher biosafety lab and can be performed in a high-throughput manner. For proof of concept, we demonstrated this method to discover novel inhibitors against SARS-CoV-2. We believe the proposed method, in combination with available drug libraries, may accelerate the identification of novel antivirals.