Transferase-mediated dUTP Nick-end Labeling Assay Research Articles

IGF2/IGFBP4 reduces apoptosis and increases free cholesterol of chicken granulosa cells in vitro

Follicle selection, a crucial step in maintaining continuous egg production in chickens, is a process that relies on granulosa cells (GCs). In this study, we aimed to identify the key genes that are involved in follicle selection from our previous single-cell transcriptomic data. We used a combination of techniques and assays, including quantitative real-time PCR, immunofluorescence, Oil Red O staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), monodansylcadaverine (MDC) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, cell counting Kit-8 (CCK-8) assay, and 5-ethynyl-2-deoxyuridine (EdU) assay. Multiple indices, such as cell proliferation, cell differentiation, progesterone synthesis, lipid droplet production, total and free cholesterol content, apoptosis, and autophagy, were measured to determine the states of GCs in vitro. The results demonstrated that overexpression of genes related to insulin-like growth factor 2 (IGF2) or IGF-binding protein 4 (IGFBP4) increases intracellular free cholesterol (progesterone precursors) and lipid droplet production, inhibits apoptosis through increased autophagy, and inhibits cell proliferation. This indicates that IGF2 or IGFBP4 can maintain the survival state and improve differentiation tendency of chicken granulosa cells in vitro. Therefore, this study provides new evidence on the functions of IGFs and IGFBPs in chickens, establishing a crucial experimental foundation for understanding the regulatory mechanisms of follicle selection. In addition, our study contributes to understanding follicular development and improves the egg-laying performance of chickens.

Repurposing Valrubicin as a Potent Inhibitor of Ovarian Cancer Cell Growth.

Ovarian cancer (OC) is a leading cause of cancer-related mortality among women, and there remains a significant unmet need for new therapeutic agents to improve patient outcomes. This study aimed to explore drug repositioning by screening a library of Food and Drug Administration (FDA)-approved compounds to identify those with therapeutic potential against OC. We also aimed to elucidate the molecular mechanisms of action of such compounds to better understand how they inhibit cancer cell proliferation. Using the WST-1 assay, a library of 1710 FDA-approved drugs was screened to evaluate their effects on OC cell proliferation. The molecular mechanisms underlying the effects of selected compounds were assessed through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunoblot analysis. Screening of FDA-approved libraries revealed valrubicin as a potent inhibitor of OVCAR8 cell proliferation and SKOV3 and A2780 cell growth. Furthermore, valrubicin treatment led to increased DNA fragmentation, as evidenced by the TUNEL assay, and activated apoptosis signaling through enhancement of cleaved caspase-3 and poly(ADP-ribose) polymerase levels. Valrubicin, through drug repositioning, can be applied as a new therapeutic agent for OC.

Recryopreservation impairs blastocyst implantation potential via activated endoplasmic reticulum stress pathway and induced apoptosis.

Recryopreservation (recryo) is occasionally applied in clinical, while the underlying mechanism of impaired clinical outcomes after recryo remains unclear. In this study, frozen embryo transfer (FET) cycles of single blastocyst transfer in an academic reproductive medicine center were enrolled. According to the number of times blastocysts experienced cryopreservation, they were divided into the cryopreservation (Cryo) group and the Recryo group. Donated human blastocysts were collected and detected for mechanism exploration. It was found that recryo procedure resulted in impaired blastocyst developmental potential, including decreased implantation rate, reduced biochemical pregnancy rate, declined clinical pregnancy rate, higher early miscarriage rate, and lower live birth rate. Moreover, recryo led to impaired trophectoderm (TE) function, exhibiting lower human chorionic gonadotropin levels 12 days after FET. In addition, single-cell RNA sequencing showed that the expression of genes involved in cell adhesion and embryo development were altered. More specifically, activated endoplasmic reticulum (ER) pathway and induced apoptosis were further verified by immunofluorescence and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay involving in the recryo procedure. In conclusion, recryo could interfere with the process of blastocyst implantation by impairing TE function, affecting blastocyst adhesion, activating ER stress pathway and inducing apoptosis. It provides caution to embryologists about the potential risk of recryopreservation.

Aucubin Alleviates Chronic Obstructive Pulmonary Disease by Activating Nrf2/HO-1 Signaling Pathway.

Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease with high death rates. Aucubin is an iridoid glycoside extracted from Eucommia ulmoides with antioxidative and anti-inflammatory properties in human diseases. This study aimed to investigate its specific function in mouse and cell models of COPD. The COPD mouse model was established by exposing mice to a long-term cigarette smoke (CS). The number of inflammatory cells and the contents of inflammatory factors tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-8 in bronchoalveolar lavage fluid (BALF) of CS-exposed mice were measured. The levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and myeloperoxidase (MPO) in the lung tissues were estimated. Masson staining and hematoxylin-eosin (H&E) staining were utilized to evaluate pulmonary fibrosis and emphysema in CS-treated mice. Cell apoptosis in the lung tissues was estimated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Western blot was applied to quantify protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and apoptotic markers. COPD cell model was established by exposing mouse lung epithelial cells (MLE12) with cigarette smoke extract to further verify the properties of aucubin in vitro. Aucubin reduced the number of inflammatory cells and decreased the contents of TNF-α, IL-6, and IL-8 in BALF of CS-treated mice. The oxidative stress, lung emphysema, fibrosis, and lung cell apoptosis induced by CS exposure were ameliorated by aucubin administration. Aucubin activated the Nrf2/HO-1 signaling pathway in vitro and in vivo. Pretreatment with ML385, a specific Nrf2 inhibitor, antagonized the protective effects of aucubin on inflammation, oxidative stress, fibrosis, and cell apoptosis in COPD. Aucubin alleviates inflammation, oxidative stress, apoptosis, and pulmonary fibrosis in COPD mice and CSE-treated MLE12 cells by activating the Nrf2/HO-1 signaling pathway.

Integrated transcriptomic and metabolomic analysis provides insight into the pollen development of CMS-D1 rice

BackgroundCytoplasmic male sterility (CMS) has greatly improved the utilization of heterosis in crops due to the absence of functional male gametophyte. The newly developed sporophytic D1 type CMS (CMS-D1) rice exhibits unique characteristics compared to the well-known sporophytic CMS-WA line, making it a valuable resource for rice breeding.ResultsIn this research, a novel CMS-D1 line named Xingye A (XYA) was established, characterized by small, transparent, and shriveled anthers. Histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays conducted on anthers from XYA and its maintainer line XYB revealed that male sterility in XYA is a result of delayed degradation of tapetal cells and abnormal programmed cell death (PCD) of microspores. Transcriptome analysis of young panicles revealed that differentially expressed genes (DEGs) in XYA, compared to XYB, were significantly enriched in processes related to chromatin structure and nucleosomes during the microspore mother cell (MMC) stage. Conversely, processes associated with sporopollenin biosynthesis, pollen exine formation, chitinase activity, and pollen wall assembly were enriched during the meiosis stage. Metabolome analysis identified 176 specific differentially accumulated metabolites (DAMs) during the meiosis stage, enriched in pathways such as α-linoleic acid metabolism, flavone and flavonol biosynthesis, and linolenic acid metabolism. Integration of transcriptomic and metabolomic data underscored the jasmonic acid (JA) biosynthesis pathway was significant enriched in XYA during the meiosis stage compared to XYB. Furthermore, levels of JA, MeJA, OPC4, OPDA, and JA-Ile were all higher in XYA than in XYB at the meiosis stage.ConclusionsThese findings emphasize the involvement of the JA biosynthetic pathway in pollen development in the CMS-D1 line, providing a foundation for further exploration of the molecular mechanisms involved in CMS-D1 sterility.

Phenethyl isothiocyanate inhibits the carcinogenic properties of hepatocellular carcinoma Huh7.5.1 cells by activating MAPK/PI3K-Akt/p53 signaling pathways.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with limited effective treatment options. Phenethyl isothiocyanate (PEITC) is a bioactive substance present primarily in the cruciferous vegetables. PEITC has exhibited anti-cancer properties in various cancers, including lung, bile duct, and prostate cancers. It has been demonstrated that PEITC can inhibit the proliferation, invasion, and metastasis of SK-Hep1 cells, while effectively inducing apoptosis and cell cycle arrest in HepG2 cells. However, knowledge of its anti-carcinogenic effects on Huh7.5.1 cells and its underlying mechanism remains elusive. In the present study, we aim to evaluate the anti-carcinogenic effects of PEITC on human HCC Huh7.5.1 cells. MTT assay and colony formation assay was performed to investigate the anti-proliferative effects of PEITC against Huh7.5.1 cells. The pro-apoptosis effects of PEITC were determined by Annexin V-FITC/PI double staining assay by flow cytometry (FCM), mitochondrial transmembrane potential (MMP) measurement, and Caspase-3 activity detection. A DAPI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was conducted to estimate the DNA damage in Huh7.5.1 cells induced by PEITC. Cell cycle progression was determined by FCM. Transwell invasion assay and wound healing migration assay were performed to investigate the impact of PEITC on the migration and invasion of Huh7.5.1 cells. In addition, transcriptome sequencing and gene set enrichment analysis (GSEA) were used to explore the potential molecular mechanisms of the inhibitory effects of PEITC on HCC. Quantitative real-time PCR (qRT-PCR) analysis was performed to verify the transcriptome data. MTT assay showed that treatment of Huh7.5.1 cells with PEITC resulted in a dose-dependent decrease in viability, and colony formation assay further confirmed its anti-proliferative effect. Furthermore, we found that PEITC could induce mitochondrial-related apoptotic responses, including a decrease of mitochondrial transmembrane potential, activation of Caspase-3 activity, and generation of intracellular reactive oxygen species. It was also observed that PEITC caused DNA damage and cell cycle arrest in the S-phase in Huh7.5.1 cells. In addition, the inhibitory effect of PEITC on the migration and invasion ability of Huh7.5.1 cells was assessed. Transcriptome sequencing analysis further suggested that PEITC could activate the typical MAPK, PI3K-Akt, and p53 signaling pathways, revealing the potential mechanism of PEITC in inhibiting the carcinogenic properties of Huh7.5.1 cells. PEITC exhibits anti-carcinogenic activities against human HCC Huh7.5.1 cells by activating MAPK/PI3K-Akt/p53 signaling pathways. Our results suggest that PEITC may be useful for the anti-HCC treatment.

Beneficial effects of human umbilical cord mesenchymal stem cell (HUCMSC) transplantation on cyclophosphamide (CTX)-induced premature ovarian failure (POF) in Tibetan miniature pigs

BackgroundPremature ovarian failure (POF), also known as primary ovarian insufficiency, is a common endocrine disease in young women. The emergence of regenerative medicine using stem cells may improve ovarian function and structure, and represents a promising prospect for POF treatment. In his study, we explored the therapeutic effects of human umbilical cord mesenchymal stem cell (HUCMSC) transplantation in a Tibetan miniature pig model of cyclophosphamide (CTX)-induced POF. MethodsWe cultured and identified HUCMSCs, labeled them with DiR iodide red dye, and implanted them into a CTX-induced model of POF in Tibetan miniature pigs. The daily weight changes were recorded, and the levels of estradiol (E2) and follicle-stimulating hormone (FSH) were measured on days 0, 7, and 14. At the end of the 21-day observation period, in vivo imaging of the bilateral ovaries was performed, and the ovarian index was measured. Ovarian tissue morphology and follicles were examined by hematoxylin-eosin staining. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was employed to assess cell apoptosis, and immunohistochemistry was used to determine the levels of p-AKT, p-ERK1/2, BAX, and BCL2 expression. ResultsOur analysis indicated successful delivery of HUCMSCs to the ovaries of the POF pig model. Significant increases were observed in body weight, E2 levels, ovarian index, and number of normal follicles (all p < 0.05). Moreover, FSH levels reduced and ovarian tissue morphology improved following HUCMSCs transplantation (all p < 0.05). Importantly, upregulated p-AKT, p-ERK1/2, and BCL2 expression were observed, whereas the expression of BAX was suppressed (all p < 0.05), suggesting the inhibition of ovarian cell apoptosis. ConclusionOur study highlights the significant therapeutic effects of HUCMSC transplantation on CTX-induced POF in a Tibetan miniature pig model.

Infantile Hemangiomas Lose Vascular Endothelial Cadherin During Involution: Potential Role in Cell Death?

Background: Infantile hemangiomas (IHs) are benign endothelial cell (EC) tumors that undergo a predictable natural history, with rapid proliferation, stabilization, and involution. However, mechanisms regulating these transitions are not well understood. We have observed loss of vascular endothelial cadherin (VECAD) in involuting/involuted IHs. VECAD plays a critical role in angiogenesis, cell cycle progression, and EC survival. We hypothesize that loss of VECAD is associated with apoptosis occurring during IH involution. Methods: Resected IH samples were clinically categorized as proliferating (n = 4), stable (n = 4), or involuting/involuted (n = 5). Neonatal dermal tissues were used as controls (n = 5). Immunohistochemistry was conducted on sectioned specimens using antibodies against EC markers VECAD and CD31. Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Results: CD31 signal intensity in proliferating, stable, and involuting/involuted IH ECs was unchanged relative to each other and to control ECs. VECAD signal significantly and progressively diminished as IHs progressed from proliferation to involution. Involuting/involuted IHs had significantly reduced VECAD expression compared with control ECs (P &lt; 0.0001), proliferating IHs (P &lt; 0.0001), and stable IHs (P &lt; 0.001). As expected, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive ECs was significantly higher in involuting/involuted IHs (P &lt; 0.05) relative to control ECs and proliferating IHs. Conclusions: Loss of VECAD expression in IH endothelium corresponded to IH involution and increased apoptosis. It is unclear whether loss of VECAD is causative of IH involution; further studies are needed to elucidate the role of VECAD function in EC survival.

Vaccine Development for Severe Fever with Thrombocytopenia Syndrome Virus in Dogs.

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening viral zoonosis. The causative agent of this disease is the Dabie bandavirus, which is usually known as the SFTS virus (SFTSV). Although the role of vertebrates in SFTSV transmission to humans remains uncertain, some reports have suggested that dogs could potentially transmit SFTSV to humans. Consequently, preventive measures against SFTSV in dogs are urgently needed. In the present study, dogs were immunized three times at two-week intervals with formaldehyde-inactivated SFTSV with two types of adjuvants. SFTSV (KCD46) was injected into all dogs two weeks after the final immunization. Control dogs showed viremia from 2 to 4days post infection (dpi), and displayed white pulp atrophy in the spleen, along with a high level of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay (TUNEL) positive area. However, the inactivated SFTSV vaccine groups exhibited rare pathological changes and significantly reduced TUNEL positive areas in the spleen. Furthermore, SFTSV viral loads were not detected at any of the tested dpi. Our results indicate that both adjuvants can be safely used in combination with an inactivated SFTSV formulation to induce strong neutralizing antibodies. Inactivated SFTSV vaccines effectively prevent pathogenicity and viremia in dogs infected with SFTSV. In conclusion, our study highlighted the potential of inactivated SFTSV vaccination for SFTSV control in dogs.

Overexpression of KAT8 induces a failure in early embryonic development in mice

Embryo quality is strongly associated with subsequent embryonic developmental efficiency. However, the detailed function of lysine acetyltransferase 8 (KAT8) during early embryonic development in mice remains elusive. In this study, we reported that KAT8 played a pivotal role in the first cleavage of mouse embryos. Immunostaining results revealed that KAT8 predominantly accumulated in the nucleus throughout the entire embryonic developmental process. Kat8 overexpression (Kat8-OE) was correlated with early developmental potential of embryos to the blastocyst stage. We also found that Kat8-OE embryos showed spindle-assembly defects and chromosomal misalignment, and that Kat8-OE in embryos led to increased levels of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX by affecting the expression of critical genes related to mitochondrial respiratory chain and antioxidation pathways. Subsequently, cellular apoptosis was activated as confirmed by TUNEL (Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling) assay. Furthermore, we revealed that KAT8 was related to regulating the acetylation status of H4K16 in mouse embryos, and Kat8-OE induced the hyperacetylation of H4K16, which might be a key factor for the defective spindle/chromosome apparatus. Collectively, our data suggest that KAT8 constitutes an important regulator of spindle assembly and redox homeostasis during early embryonic development in mice.

Resveratrol Suppresses “Metabolic Memory” by Inhibiting Inflammation and Apoptosis Through the ROS/TXNIP/NLRP3 Signaling Pathway

Aim: This research endeavored to explore the impact and underlying mechanisms of resveratrol on the phenomenon of “metabolic memory” in cultured human retinal vascular endothelial cells (HRVECs) under high-glucose (HG) conditions. Materials and Methods: According to the glucose level and treatment, cultured HRVECs were divided into seven groups: normal glucose (NG), HG, high glucose followed by NG (HN), mannitol (Man), resveratrol, thioredoxin-interacting protein (TXNIP)-small interfering ribonucleic acid (siRNA), and N-acetylcysteine (NAC). The expression levels of TXNIP, nucleotide oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, intercellular adhesion molecule 1 (ICAM-1), caspase-1, interleukin-1β (IL-1β), B-cell lymphoma 2 (Bcl-2), caspase-3, and Bcl-2-associated X (BAX), as well as reactive oxygen species (ROS) production, were measured. Cell apoptosis was assessed through a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Results: In HRVECs from the HG group, expression levels of TXNIP, NLRP3, caspase-1, ICAM-1, and IL-1β were upregulated. However, in the HN group, the above upregulations were not reversed. After the administration of resveratrol, the expression levels of TXNIP, NLRP3, and other inflammatory cytokines were significantly reduced. Resveratrol mitigated the elevated ROS production induced by HG conditions. In the NAC group, the expression of TXNIP and inflammatory cytokines was downregulated. TXNIP-siRNA treatment showed similar effects. Resveratrol inhibited apoptosis as well as reversed the downregulation of BCL-2 and the upregulation of caspase-3 and BAX induced by HG conditions. Conclusion: Resveratrol mitigated the HG-induced phenomenon of “metabolic memory” by inhibiting inflammation and apoptosis via modulation of the ROS/TXNIP/NLRP3 signaling pathway in cultured HRVECs. Therefore, resveratrol may have therapeutic potential to treat diabetic retinopathy and related metabolic memory complications.

Immunoprotective effect of silybin through blocking p53-driven caspase-9-Apaf-1-Cyt c complex formation and immune dysfunction after difenoconazole exposure in carp spleen.

As a broad-spectrum and efficient triazole fungicide, difenoconazole is widely used, which not only pollutes the environment but also exerts toxic effects on non-target organisms. The spleen plays an important role in immune protection as an important secondary lymphoid organ in carp. In this study, we assessed the protective impact of silybin as a dietary additive on spleen tissues of carp during exposure to difenoconazole. Sixty carp were separated into four groups for this investigation including control group, difenoconazole group, silybin group, and silybin and difenoconazole group. By hematoxylin-eosin staining, dihydroethidium staining, immunohistochemical staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, quantitative real-time PCR assay, Western blot analysis, biochemical assays, and immune function indicator assays, we found that silybin could prevent difenoconazole-induced spleen tissue damage, oxidative stress, and immune dysfunction, and inhibited apoptosis of carp spleen tissue cells by suppressing the formation of p53-driven caspase-9-apoptotic protease activating factor-1-cytochrome C complex. The results suggested that silybin as a dietary additive could improve spleen tissue damage and immune dysfunction induced by difenoconazole in aquaculture carp.

LncRNA PART1 Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating TFAP2C/DUSP5 Axis via miR-302a-3p.

Myocardial ischemia-reperfusion injury (MIRI) refers to the damage of cardiac function caused by restoration of blood flow perfusion in ischemic myocardium. However, long non-coding RNA prostate androgen regulated transcript 1 (PART1)'s role in MIRI remain unclear. Immunofluorescence detected LC3 expression. Intermolecular relationships were verified by dual luciferase reporter assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and transferase-mediated dUTP nick-end labeling (TUNEL) assays analyzed cell viability and apoptosis. The release of lactate dehydrogenase was tested via enzyme-linked immunosorbent assay (ELISA). Left anterior descending coronary artery surgery induced a MIRI mouse model. Infarct area was detected by 2,3,5-triphenyltetrazolium chloride staining. Hematoxylin and eosin staining examined myocardial injury. ELISA evaluated myocardial marker (creatine kinase MB) level. PART1 was decreased in hypoxia/reoxygenation (H/R) induced AC16 cells and MIRI mice. PART1 upregulation attenuated the increased levels of Bax, beclin-1 and the ratio of LC3II/I, and enhanced the decrease of Bcl-2 and p62 expression in H/R-treated cells. PART1 upregulation alleviated H/R-triggered autophagy and apoptosis via miR-302a-3p. Mechanically, PART1 targeted miR-302a-3p to upregulate transcription factor activating enhancer-binding protein 2C (TFAP2C). TFAP2C silencing reversed the protected effects of miR-302a-3p inhibitor on H/R treated AC16 cells. We further established TFAP2C combined to dual-specificity phosphatase 5 (DUSP5) promoter and activated DUSP5. TFAP2C upregulation suppressed H/R-stimulated autophagy and apoptosis through upregulating DUSP5. Overexpressed PART1 reduced myocardial infarction area and attenuated MIRI in mice. PART1 improved the autophagy and apoptosis in H/R-exposed AC16 cells through miR-302a-3p/TFAP2C/DUSP5 axis, which might provide novel targets for MIRI treatment.

Medicinal and edible plant Allium macrostemon Bunge for the treatment of testosterone-induced androgenetic alopecia in mice

Ethnopharmacological relevanceAllium macrostemon Bunge (AMB), a widely distributed wild garlic plant, possesses a variety of health-promoting properties. Androgenetic alopecia (AGA) is a common disorder that affects quality of life. Aim of the studyWe sought to investigate whether AMB stimulates hair regrowth in AGA mouse model, and clarify the underlying molecular mechanisms. Materials and methodsThe chemical constituents of AMB water extract were identified by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q/TOF-MS) analysis. Cell viability assay and Ki-67 immunostaining were undertaken to evaluate the impacts of AMB on human hair dermal papilla cell (HDPC) proliferation. Wound-healing assay was undertaken to assess cell migration. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were performed to examine cell apoptosis. Western blotting, real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and immunostaining assays were undertaken to determine the impacts of AMB on Wnt/β-catenin signaling and growth factors expression in HDPC cells. AGA mouse model was induced by testosterone treatment. The effects of AMB on hair regeneration in AGA mice were demonstrated by hair growth measuring and histological scoring. The levels of β-catenin, p-GSK-3β, and Cyclin D1 in dorsal skin were measured. ResultsAMB promoted proliferation and migration, as well as the expression of growth factors in cultured HDPC cells. Meanwhile, AMB restrained apoptosis of HDPC cells by increasing the ratio of anti-apoptotic Bcl-2/pro-apoptotic Bax. Besides, AMB activated Wnt/β-catenin signaling and thereby enhancing growth factors expression as well as proliferation of HDPC cells, which was abolished by Wnt signaling inhibitor ICG-001. In addition, an increase of hair shaft elongation was observed in mice suffering from testosterone-induced AGA upon the treatment of AMB extract (1% and 3%). Consistent with the in vitro assays, AMB upregulated the Wnt/β-catenin signaling molecules in dorsal skin of AGA mice. ConclusionThis study demonstrated that AMB promoted HDPC cell proliferation and stimulated hair regrowth in AGA mice. Wnt/β-catenin signaling activation, which induced production of growth factors in hair follicles and, eventually, contributed to the influence of AMB on the hair regrowth. Our findings may contribute to effective utilization of AMB in alopecia treatment.

Profilin1 Regulates Trophoblast Invasion and Macrophage Differentiation

Unexplained recurrent spontaneous abortion (URSA) has been associated with the dysfunction of trophoblasts and decidual macrophages. Current evidence suggests that profilin1 (PFN1) plays an important role in many biological processes. However, little is known about whether PFN1 is related to URSA. Herein, the location of PFN1 was detected by immunohistochemistry, and the level of PFN1 was detected by quantitative real-time PCR, Western blot analysis, and immunohistochemistry. The proliferation of trophoblasts was detected by CCK8 and 5-ethynyl-2'-deoxyuridine assays, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were used to detect apoptosis of trophoblasts. The migration and invasion ability of trophoblasts was assessed by using the wound-healing test and transwell test. Polarization of macrophages was detected in macrophages cultured in trophoblast conditioned medium. PFN1 expression was observed in cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts and was decreased in the villous tissue of patients with URSA. The migration and invasion ability and cell viability of trophoblastic cell lines that underwent PFN1 knockdown significantly decreased, and apoptosis increased. Opposite findings were observed after the overexpression of PFN1 in trophoblastic cells. In addition, PFN1 could regulate trophoblast function through phosphatidylinositol 3-kinase/AKT signal transduction rather than mitogen-activated proteinkinasesignaling pathways. Finally, knockdown of PFN1 in trophoblasts promoted tumor necrosis factor-α secretion to induce macrophage polarization to M1 phenotype, mediated by the NF-κB signaling pathway. These findings indicate that PFN1 has a broad therapeutic potential for patients with URSA.

Eriocalyxin B inhibits inflammation induced by CCI-induced microglia activation to relieve neuropathic pain through inhibition of JAK2/STAT3 and NF-κB pathways

Neuropathic pain is a very troublesome disease that seriously affects human life. Eriocalyxin B (EriB) has been revealed to attenuate various diseases through its anti-inflammatory effects, but its regulatory effects on neuropathic pain remains unclear. The paw withdrawal threshold and paw withdrawal thermal latency were detected through mechanical allodynia and thermal hyperalgesia tests. The spinal injury was assessed through hematoxylin and eosin staining. The cell apoptosis was measured through terminal deoxynucleotide transferase-mediated dUTP nick end-labeling assay. The protein expressions were examined through Western blot analysis. The mRNA expression was examined through reverse transcription-quantitative polymerase chain reaction. The ionized calcium-binding adaptor molecule 1 level in the spinal cord was evaluated through immunofluorescence assay. The levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were measured through enzyme-linked-immunosorbent serologic assay. The chronic constriction injury (CCI) rat model was constructed for the study. Our results demonstrated that EriB relieved CCI-stimulated neuropathic pain and nerve damage. In addition, the enhanced neural apoptosis mediated by CCI induction was reduced after EriB treatment. In addition, EriB inhibited CCI-induced microglia activity and inflammation. At last, the Janus kinase 2–signal transducer and activator of transcription 3 (JAK2/STAT3) and nuclear factor kappa B (NF-κB) pathways were activated in CCI rat model, which were attenuated following EriB treatment. Importantly, EriB (10 mg/kg) had a strong effect that was similar to the positive control (1-μg/kg dexmedetomidine), suggesting that EriB may be an effective drug for neuropathic pain. This study demonstrated that EriB inhibited inflammation caused by CCI-induced microglia activation to relieve neuropathic pain through inhibition of JAK2/STAT3 and NF-κB pathways. This study may highlight the regulatory functions of EriB in the treatment of neuropathic pain.

Rice tapetum differentiation is sensitive to downregulation of OsUCH3, a ubiquitin C-terminal hydrolase.

The tapetum is a special tissue within the stamen that supplies nutrients to support microspore differentiation. Many genes have been identified to function in tapetum differentiation (Astrand et al., 2021). Ubiquitin C-terminal hydrolase (UCH) is a type of cysteine protease involved in protein deubiquitination process (Mevissen and Komander, 2017) and plays roles in gonadal transformation and spermatogenesis in animals (Luo et al., 2009; Kwon et al,. 2004; Wang et al., 2006). OsUCH3 (Os02g43760) was highly expressed in early rice stamen cells (Chen et al., 2015), the OsUCH3 expression was first observed in all cells of anther primordium and then gradually concentrated into the germ cells and tapetum cells, and OsUCH3 protein exhibits stronger ubiquitin C-terminal hydrolase enzymatic activity (Wang et al., 2018). These data suggested that OsUCH3 is likely associated with stamen development. To explore whether OsUCH3 plays roles in rice stamen development, we constructed transgenic lines to down-regulate OsUCH3 expression through RNAi vector as described previously with primers (ccACTAGTATGGAGGATGCTCATTCC and TcGGATCCCACAACTTTCGAAAGAGC) (Liu et al., 2005) (Figure 1a–g) and the OsUCH3-targeting CRISPR-Cas9 mutant lines (Figure 1h). CRISPR-Cas9 osuch3 mutant lines exhibited the same male-sterile phenotype as OsUCH3R. In observation of the overall morphology, there was no significant difference at vegetable development (Figure 1a). However, low seed setting rate was observed in the RNAi lines (Figure 1b) and CRISPR-Cas9 osuch3 mutant (Figure 1h). Alexander staining showed that OsUCH3R lines had abnormal pollen (Figure 1d) with the latter being indicated by the significant reduction in pollen grain number (Figure 1e, g). The RT-qPCR results revealed that OsUCH3 expression levels were significantly reduced in OsUCH3R transgenic rice (Figure 1f). To examine whether OsUCH3 affected stamen development, we pollinated OsUCH3R pistils with wild-type pollen grains, which rescued the sterility of OsUCH3R. The result suggested that the low seed setting rate resulted mainly from down-expression of OsUCH3 (Figure 1i). Morphological analysis revealed abnormal tapetum differentiation and locule collapse were observed in the OsUCH3R lines from stage 5 (S5) through 12 (S12) (Figure 1j). This observation suggested that the downregulation of OsUCH3 caused a defect in the tapetum differentiation and stamen development. In contrast to the degeneration of the tapetum that was observed in the WT from stages 7 (S7) to 9 (S9) (Figure 1k–m), the tapetum in the OsUCH3R lines was not properly degenerated during these stages (Figure 1n–p) as revealed by transmission electron microscopy (TEM) analysis. In addition, TEM observations revealed that the secondary cell walls of the endothecium cells were thinner in the OsUCH3R lines than in the wild type (Figure 1p, red arrowhead). We found green fluorescence in wild-type tapetal cells from stages 7 to 9 (Figure 1q-t) using the terminal deoxynucleotidyl transferase-mediated TdT-mediated dUTP Nick-End Labeling (TUNEL) assay (Chen et al., 2018). No such TUNEL signals were detected in OsUCH3R tapetal cells during the same developmental period (Figure 1u–x). These results suggested that DNA degradation during tapetum differentiation after stage 7 was abnormal in OsUCH3R. Numerous genes were known being involved in tapetum differentiation (Chen et al., 2018). GAMYB4 was up-regulated while the expression of YY1, OsCP1 and UDT1 exhibited no change in the OsUCH3R lines (Figure 1y). OsUCH3 is lower expressed in shoot tips and leaf primordia. While no differential expression of SAM genes OsWOX3, the expression of OSH1 and OsWUS was significantly decreased in the OsUCH3R lines (Figure 1y). These changes demonstrated the link between abnormal tapetum differentiation in the OsUCH3R lines with the changes in the expression levels of known tapetum genes. The phenotypes we found in OsUCH3R lines and CRISPR mutants demonstrated that tapetal differentiation is sensitive to the change of expression level of OsUCH3 and protein deubiquitination is an importance factor for proper tapetum differentiation. This finding provided a new opportunity not only in deciphering the regulatory network of tapetum differentiation at protein level, but in manipulating male sterility for agricultural applications. Sequence data from this article can be found in the GenBank/EMBL data library. This work was supported by grants from the Chinese Transgenic Project (grant no. CARS-01-06, 2016ZX08010001 and 2016ZX08009-003) and the National Natural Science Foundation of China (grant no. 31630006). The authors declare no competing interests. D.H.W., Z.H.X. and S.N.B. designed the research and wrote the manuscript. D.H.W. performed most of the experiments. N.L. and S.D.Y. provided an assistance for mutant genotyping and plant material fixation. Z.S.C and Y.N.L provided technical assistance for transgenic plant analyse. Z.H.L helped the semithin sectioning.

Lithium Chloride Exerts Anti-Inflammatory and Neuroprotective Effects by Inhibiting Microglial Activation in LPS-Induced Retinal Injury.

To explore the anti-inflammatory and neuroprotective effects of lithium chloride (LiCl) in LPS-induced retinal injury. In vitro, primary retinal microglia were pretreated with LiCl and stimulated with lipopolysaccharide (LPS). Pro-inflammatory cytokine production, microglial morphological changes, and inflammation-associated signaling pathways were measured by real-time PCR (RT-PCR), western blotting, and immunofluorescence. Primary retinal neurons were cultured with microglial-derived conditioned medium in the absence or presence of LiCl. Neurotoxicity was evaluated by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and γ-H2AX detection. In vivo, an endotoxin-induced uveitis mice model was established, and each animal was given intraperitoneal injection of LiCl or vehicle. The retinal inflammatory response was measured by hematoxylin and eosin and fluorescent staining, RT-PCR, western blotting, and TUNEL assay. Retinal thickness and function were evaluated by spectral-domain optical coherence tomography and electroretinography. In vitro, LiCl exerted no obvious toxic effects on microglia and significantly decreased proinflammatory factor (inducible nitric oxide synthase, tumor necrosis factor α, interleukin 6) production, inhibited microglial activation in morphology, and suppressed nuclear factor kappa B (NF-κB), Akt, and phosphatidylinositol 3-kinase (PI3K) phosphorylation. Moreover, LiCl promoted retinal neuron survival and reduced cell apoptosis and the expression of γ-H2AX. In vivo, LiCl reduced inflammatory infiltrating cells in the vitreous cavity and decreased proinflammatory cytokine expression in retinas. LiCl suppressed LPS-induced microglial activation, proliferation, and migration. Additionally, LiCl reduced LPS-induced apoptosis of ganglion cells and retinal edema and rescued retinal functional damage. This study demonstrates that LiCl exerts anti-inflammatory and neuroprotective effects by inhibiting microglial activation via the PI3K/Akt/NF-κB pathway in LPS-induced retinal injury. LiCl provides a novel and promising option to treat retinal inflammatory diseases.

Mechanism Repositioning Based on Integrative Pharmacology: Anti-Inflammatory Effect of Safflower in Myocardial Ischemia–Reperfusion Injury

Safflower (Carthamus tinctorius. L) possesses anti-tumor, anti-thrombotic, anti-oxidative, immunoregulatory, and cardio-cerebral protective effects. It is used clinically for the treatment of cardio-cerebrovascular disease in China. This study aimed to investigate the effects and mechanisms of action of safflower extract on myocardial ischemia-reperfusion (MIR) injury in a left anterior descending (LAD)-ligated model based on integrative pharmacology study and ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometer (UPLC-QTOF-MS/MS). Safflower (62.5, 125, 250 mg/kg) was administered immediately before reperfusion. Triphenyl tetrazolium chloride (TTC)/Evans blue, echocardiography, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, lactate dehydrogenase (LDH) ability, and superoxide dismutase (SOD) levels were determined after 24 h of reperfusion. Chemical components were obtained using UPLC-QTOF-MS/MS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze mRNA and protein levels, respectively. Safflower dose-dependently reduced myocardial infarct size, improved cardiac function, decreased LDH levels, and increased SOD levels in C57/BL6 mice. A total of 11 key components and 31 hub targets were filtered based on the network analysis. Comprehensive analysis indicated that safflower alleviated inflammatory effects by downregulating the expression of NFκB1, IL-6, IL-1β, IL-18, TNFα, and MCP-1 and upregulating NFκBia, and markedly increased the expression of phosphorylated PI3K, AKT, PKC, and ERK/2, HIF1α, VEGFA, and BCL2, and decreased the level of BAX and phosphorylated p65. Safflower shows a significant cardioprotective effect by activating multiple inflammation-related signaling pathways, including the NFκB, HIF-1α, MAPK, TNF, and PI3K/AKT signaling pathways. These findings provide valuable insights into the clinical applications of safflower.

Zhao’s Weitiao No 3 inhibits tumor growth in gastric cancer-bearing nude mice

Purpose: To investigate the effect of Zhao’s Weitiao No. 3 (ZW3) on the progression of gastric cancer (GC) in mice, and to determine its effects on the proliferation and apoptosis of gastric cancer tissues.&#x0D; Methods: Tumor growth in vivo was used to determine the effect of ZW3 (15, 30, and 60 mg/kg) on GC growth in mice. Immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were conducted to evaluate the effects of ZW3 on the proliferation and apoptosis of mouse GC tissues, while quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunoblot assays were conducted to determine its effects on GC cell apoptosis.&#x0D; Results: ZW3 inhibited tumor growth in tumor-bearing mice, suppressed the expression of proliferation-related proteins (p &lt; 0.001), increased the apoptosis of tumor cells in tumor-bearing mice (p &lt; 0.001), and suppressed the expression of apoptosis-related proteins in mouse tumor tissues (p &lt; 0.001).&#x0D; Conclusion: Thus, ZW3 is a potential drug for the management of GC. However, further investigations are required to determine its full potential