Relative Protein Expression Levels Research Articles

CDC45 promotes the stemness and metastasis in lung adenocarcinoma by affecting the cell cycle

ObjectiveThis study aimed to assess the functions of cell division cycle protein 45 (CDC45) in Non-small cell lung cancer (NSCLC) cancer and its effects on stemness and metastasis.MethodsFirstly, differentially expressed genes related to lung cancer metastasis and stemness were screened by differential analysis and lasso regression. Then, in vitro, experiments such as colony formation assay, scratch assay, and transwell assay were conducted to evaluate the impact of CDC45 knockdown on the proliferation and migration abilities of lung cancer cells. Western blotting was used to measure the expression levels of related proteins and investigate the regulation of CDC45 on the cell cycle. Finally, in vivo model with subcutaneous injection of lung cancer cells was performed to verify the effect of CDC45 on tumor growth.ResultsThis study identified CDC45 as a key gene potentially influencing tumor stemness and lymph node metastasis. Knockdown of CDC45 not only suppressed the proliferation and migration abilities of lung cancer cells but also caused cell cycle arrest at the G2/M phase. Further analysis revealed a negative correlation between CDC45 and cell cycle-related proteins, stemness-related markers, and tumor mutations. Mouse experiments confirmed that CDC45 knockdown inhibited tumor growth.ConclusionAs a novel regulator of stemness, CDC45 plays a role in regulating lung cancer cell proliferation, migration, and cell cycle. Therefore, CDC45 may serve as a potential target for lung cancer treatment and provide a reference for further mechanistic research and therapeutic development.

Ivacaftor attenuates gentamicin-induced ototoxicity through the CFTR-Nrf2-HO1/NQO1 pathway

ABSTRACT Objectives Gentamicin is one of the most common ototoxic drugs that can lower patients' quality of life. Oxidative stress is a key factors inducing sensory hair cell death during gentamicin administration. So far, there are no effective drugs to prevent or treat gentamicin- induced hearing loss. A recent study found cystic fibrosis transmembrane conductance regulator (CFTR) as a new target to modulate cellular oxidative balance. The objective of this study was to estimate the effect of the CFTR activator ivacaftor on gentamicin-induced ototoxicity and determine its mechanism. Methods The hair cell count was analyzed by Myosin 7a staining. Apoptosis was analyzed by TUNEL Apoptosis Kit. Cellular reactive oxygen species (ROS) level was detected by DCFH-DA probes. The Nrf2 related proteins expression levels were analyzed by western blot. Results An in vitro cochlear explant model showed that gentamicin caused ROS accumulation in sensory hair cells and induced apoptosis, and this effect was alleviated by pretreatment with ivacaftor. Western blotting showed that ivacaftor administration markedly increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO1), and NAD(P)H:quinone oxidoreductase 1 (NQO1). The protective effect of ivacaftor was abolished by the Nrf2 inhibitor ML385. Discussion Our results indicate the protective role of the CFTR-Nrf2-HO1/NQO1 pathway in gentamicin-induced ototoxicity. Ivacaftor may be repositioned or repurposed towards aminoglycosides-induced hearing loss.

Human Mesenchymal Stem Cells Improve Angiogenesis and Bone Formation in Severed Finger Rats through SIRT1/Nrf2 Signaling.

This study employed a severed finger rat model to analyze the effects of human mesenchymal stem cells (MSCs) on angiogenesis, inflammatory response, apoptosis, and oxidative stress, to evaluate the possible mechanism of the repair effect of MSCs on severed finger (SF) rats. Sixty Sprague-Dawley (SD) rats were categorized into five groups (n = 12). The pathological changes of severed finger tissues were investigated by Hematoxylin and eosin (H&E) staining on day 14 after the rats were sacrificed. The levels of inflammatory factors and oxidative stress factors were detected by ELISA. Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick End Labeling (TUNEL) was employed to assess the apoptosis of chondrocytes in severed finger tissues. The expression of osteocalcin (OCN), osteopontin (OPN), Collagen I (Col-1), and CD31 were detected by immunohistochemistry or immunofluorescence assay, respectively. The expression levels of related proteins were determined by western blot. Our study presented evidence that MSCs treatment improved pathological changes of skin and bone tissue, diminished the inflammatory response, prevented oxidative stress injury, suppressed chondrocyte apoptosis, and promoted angiogenesis, and bone formation compared to the model group. In addition, EX527 treatment attenuated the effect of MSCs, SRT1720 and ML385 co-treatment also attenuated the effect of MSCs. Importantly, the MSCs treatment increased the expression of Sirtuin 1(SIRT1)/Nuclear factor erythroid2-related factor 2(Nrf2) relate proteins. Our study indicated that the mechanism of the effect of MSCs on a severed finger was related to the SIRT1/ Nrf2 signaling pathway.

Cynaroside Induces G1 Cell Cycle Arrest by Downregulating Cell Division Cycle 25A in Colorectal Cancer.

Natural chemicals derived from herbal plants have recently been recognized as potentially useful treatment alternatives owing to their ability to target a wide range of important biological molecules. Cynaroside is one of these natural compounds with promising anticancer activity for numerous tumor types. Nevertheless, the anticancer effects and molecular mechanisms of action of cynaroside on colorectal cancer (CRC) remain unclear. In this study, cynaroside was found to markedly inhibit CRC cell proliferation and colony formation in vitro. Cynaroside also inhibited cell proliferation in vivo and decreased the expression of KI67, a cell nuclear antigen. RNA sequencing revealed 144 differentially expressed genes (DEGs) in HCT116 cells and 493 DEGs in RKO cells that were enriched in the cell cycle signaling pathway. Cell division cycle 25A (CDC25A), a DEG widely enriched in the cell cycle signaling pathway, is considered a key target of cynaroside in CRC cells. Cynaroside also inhibited DNA replication and arrested cells in the G1/S phase in vitro. The expression levels of CDC25A and related G1-phase proteins were significantly elevated after CDC25A overexpression in CRC cells, which partially reversed the inhibitory effect of cynaroside on CRC cell proliferation and G1/S-phase arrest. In summary, cynaroside may be used to treat CRC as it inhibits CDC25A expression.

RFC3 drives the proliferation, migration, invasion and angiogenesis of colorectal cancer cells by binding KIF14.

Colorectal cancer (CRC) is a deadly and aggressive type of cancer that has a high fatality rate. The expression levels of replication factor C subunit 3 (RFC3) and kinesin family member 14 (KIF14) have been reported to be increased in CRC. The current study aimed to explore the effects of RFC3 on the malignant behaviors of CRC cells and its possible underlying mechanism involving KIF14. RFC3 and KIF14 expression levels in CRC tissues were analyzed using TNMplot database and Gene Expression Profiling Interactive Analysis database bioinformatics tools. RFC3 and KIF14 levels in CRC cells were examined using reverse transcription-quantitative PCR and western blotting. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were performed to assess cell proliferation. Cell apoptosis was determined using flow cytometric analysis. Wound healing and Transwell assays were adopted for the evaluation of cell migration and invasion. Tube formation assay in human umbilical vein endothelial cells was used to measure angiogenesis. Western blotting analysis was performed to determine the expression of apoptosis-, migration- and angiogenesis-associated proteins. Additionally, bioinformatics tools predicted the co-expression and interaction of RFC3 and KIF14, which was verified by a co-immunoprecipitation assay. RFC3 displayed elevated expression in CRC tissues and cells, and depletion of RFC3 halted the proliferation, migration, invasion and angiogenesis, while increasing the apoptosis of CRC cells; this was accompanied by changes in the expression levels of related proteins. In addition, RFC3 bound to KIF14 and interference with RFC3 reduced KIF14 expression. Moreover, KIF14 upregulation reversed the effects of RFC3 depletion on the aggressive cellular behaviors in CRC. In conclusion, RFC3 might interact with KIF14 to function as a contributor to the malignant development of CRC.

Docosahexaenoic Acid Modulates Nonalcoholic Fatty Liver Disease by Suppressing Endocannabinoid System.

Endocannabinoid signaling regulates energy homeostasis, and is tightly associated with nonalcoholic fatty liver disease (NAFLD). The study previously finds that supplementation of docosahexaenoic acid (DHA) has superior function to ameliorate NAFLD compared with eicosapentaenoic acid (EPA), however, the underlying mechanism remains elusive. The present study aims to investigate whether DHA intervention alleviates NAFLD via endocannabinoid system. In a case-control study, the serum endocannabinoid ligands in 60 NAFLD and 60 healthy subjects are measured. Meanwhile, NAFLD model is established in mice fed a high-fat and -cholesterol diet (HFD) for 9 weeks. DHA or EPA is administrated for additional 9 weeks. Serum primary endocannabinoid ligands, namely anandamide (AEA) and 2-arachidoniylglycerol (2-AG), are significantly higher in individuals with NAFLD compared with healthy controls. NAFLD model shows that serum 2-AG concentrations and adipocyte cannabinoid receptor 1 expression levels are significantly lower in DHA group compared with HFD group. Lipidomic and targeted ceramide analyses further confirm that endocannabinoid signaling inhibition has exerted deletion of hepatic C16:0-ceramide contents, resulting in down-regulation of de novo fatty acid synthesis and up-regulation of fatty acid β-oxidation related protein expression levels. This work elucidates that DHA has improved NAFLD by suppressing endocannabinoid system.

Sirt1 Inhibits Atrial Fibrosis by Downregulating the Expression of the Transforming Growth Factor-β1/Smad Pathway.

Atrial fibrosis is an important factor leading to atrial fibrillation, and the transforming growth factor-β1/Smad pathway is a key factor in inducing atrial fibrosis. Sirt1 is a member of the histone deacetylase (sirtuin) family, and recent studies have proven its cardioprotective effects. This study explored the effect of Sirt1 on atrial fibrosis through the transforming growth factor-β1/Smad pathway. We analyzed human right atrial appendage tissues and explored the relationship between Sirt1 and atrial fibrosis at the morphological, functional and molecular levels by Masson trichrome staining, immunofluorescence, real-time quantitative polymerase chain reaction and Western blot analysis. Rat atrial fibroblasts were extracted and treated by the Sirt1 agonist resveratrol, inhibitor sirtinol, and recombinant human transforming growth factor-β1 protein. The expression levels of related proteins were detected by Western blot, and the effect on the migration of atrial fibroblasts was detected by wound healing assay. We found that the expression of Sirt1 was reduced in the right atrial appendage tissues of patients with atrial fibrillation, and the degree of fibrosis was increased. In atrial fibroblasts, the activation of Sirt1 could inhibit the expression of transforming growth factor-β1/Smad and reduce the development of fibrosis, while inhibiting Sirt1 reduced its inhibitory effect on the transforming growth factor-β1/Smad pathway. These findings indicate that Sirt1 inhibits atrial fibrosis by downregulating the expression of the transforming growth factor-β1/Smad pathway, and provide potential targets for the treatment of atrial fibrillation.

PPP2R1B abolishes colorectal cancer liver metastasis and sensitizes Oxaliplatin by inhibiting MAPK/ERK signaling pathway

BackgroundPatients with colorectal cancer (CRC) with liver metastasis or drug resistance have a poor prognosis. Previous research has demonstrated that PPP2R1B inactivation results in the development of CRC. However, the role of PPP2R1B in CRC metastasis and drug resistance is unclear.MethodsVenny 2.1 was used to determine the intersection between survival-related differentially expressed genes (DEGs) and liver metastasis-related DEGs according to RNA-seq data from The Cancer Genome Atlas (TCGA) and the GEO database (GSE179979). LC‒MS/MS and coimmunoprecipitation were performed to predict and verify the substrate protein of PPP2R1B. Gene Set Variation Analysis (GSVA) was subsequently utilized to assess pathway enrichment levels. The predictive performance of PPP2R1B was assessed by regression analysis, Kaplan–Meier (KM) survival analysis and drug sensitivity analysis. Immunohistochemistry (IHC), qRT-PCR and western blotting were performed to measure the expression levels of related mRNAs or proteins. Biological features were evaluated by wound healing, cell migration and invasion assays and CCK-8 assays. A mouse spleen infection liver metastasis model was generated to confirm the role of PPP2R1B in the progression of liver metastasis in vivo.ResultsAccording to bioinformatics analysis, PPP2R1B is significantly associated with liver metastasis and survival in CRC patients, and these findings were further verified via immunohistochemistry (IHC), qPCR and Western blotting. Pathway enrichment and LC‒MS/MS analysis revealed that PPP2R1B is negatively associated with the MAPK/ERK signalling pathway. Additionally, PD98059, a MAPK/ERK pathway inhibitor, inhibited EMT in vitro by reversing the changes in key proteins involved in EMT signalling (ZEB1, E-cadherin and Snail) and ERK/MAPK signalling (p-ERK) mediated by PPP2R1B. Oxaliplatin sensitivity prediction and validation revealed that PPP2R1B silencing decreased Oxaliplatin chemosensitivity, and these effects were reversed by PD98059 treatment. Moreover, PPP2R1B was coimmunoprecipitated with p-ERK in vitro. A negative correlation between PPP2R1B and p-ERK expression was also observed in clinical CRC samples, and the low PPP2R1B/high p-ERK coexpression pattern indicated a poor prognosis in CRC patients. In vivo, PPP2R1B silencing significantly promoted liver metastasis.ConclusionsThis study revealed that PPP2R1B induces dephosphorylation of the p-ERK protein, inhibits liver metastasis and increases Oxaliplatin sensitivity in CRC patients and could be a potential candidate for therapeutic application in CRC.

[Impact of hyperoxia on the phenotype of pulmonary artery smooth muscle cells].

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.

Diallyl trisulfide inhibits osteosarcoma 143B cell migration, invasion and EMT by inducing autophagy

BackgroundDiallyl trisulfide (DATS), a compound derived from garlic, has been demonstrated its anti-cancer properties. While it has been shown to inhibit the expression of epidermal growth factor receptor (EGFR) in various cancers, its effects on osteosarcoma (OS) cells remain unclear. This study aimed to investigate the impacts of DATS on OS cells growth, migration, invasion, epithelial-mesenchymal transition (EMT) and autophagy, as well as its underlying mechanisms which was involving in the EGFR/PI3K/AKT/mTOR pathway. MethodsIn this study, human osteosarcoma cells (143B) were treated with different concentrations of DATS (10, 50, 100 and 200 μM) for 24 and 48 h, respectively. Cell viability was measured using CCK8, the half lethal concentration was selected for the following experiments. Wound healing and transwell assays were performed to evaluate migration and invasion abilities, while flow cytometry was used to measure apoptosis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and confocal imaging were employed to analyze the related mRNA and protein expression levels of epithelial-mesenchymal transition (EMT), EGFR/Phosphoinositide 3 kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) signaling pathway and autophagy-related markers. ResultsDATS significantly inhibited proliferation, migration and EMT in osteosarcoma cells. Additionally, DATS promoted cell apoptosis and induced autophagy, which could be rescued by the autophagy inhibitor 3-methyladenine (3-MA). Moreover, DATS treatment led to the inactivation of the EGFR/PI3K/AKT/mTOR pathway in osteosarcoma cells. ConclusionsThis study demonstrated that DATS inhibited osteosarcoma cell growth, migration and EMT, but inducing apoptosis and autophagy. These effects were mediated by the inactivation of the EGFR/PI3K/AKT/mTOR signaling pathway. These findings suggested that DATS could serve as a potential therapeutic agent for osteosarcoma treatment.

[Experimental study of tetramethylpyrazine-loaded electroconductive hydrogel on angiogenesis and neuroprotection after spinal cord injury].

To explore the mechanisms for repairing spinal cord injury (SCI) with tetramethylpyrazine-loaded electroconductive hydrogel (hereinafter referred to as "TGTP"). A total of 72 female Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (group A), SCI group (group B), SCI+electroconductive hydrogel group (group C), and SCI+TGTP group (group D). Only the vertebral plate was removed in group A, while the remaining groups were subjected to a whole transection model of spinal cord with a 2 mm gap in the lesions. The recovery of hindlimb motor function was evaluated by Basso, Beattie, Bresnahan (BBB) score and modified Rivlin-Tator inclined plate test before operation and at 1, 3, 7, 14, and 28 days after operation, respectively. Animals were sacrificed at 7 days and 28 days after modeling. Neovascularisation was observed by immunofluorescence staining of CD31 and the expression levels of angiopoietin 1 (Ang-1) and Tie-2 were assessed by Western blot assay. At 28 days postoperatively, the expression levels of pro-angiogenic related proteins, including platelet-derived growth factor B (PDGF-B), PDGF receptor β (PDGFR-β), vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2), were also assessed by Western blot. The fibrous scar in the injured area was assessed using Masson staining, while neuronal survival was observed through Nissl staining. Furthermore, LFB staining was utilized to detect myelin distribution and regeneration. Immunofluorescence and Western blot assay were employed to evaluate the expression of neurofilament 200 (NF200). The hindlimb motor function of rats in each group gradually recovered from the 3rd day after operation. The BBB score and climbing angle in group D were significantly higher than those in group B from 3 to 28 days after operation, and significantly higher than those in group C at 14 days and 28 days after operation ( P<0.05). Masson staining showed that the collagen volume fraction in groups B-D were significantly higher than that in group A, and that in group D was significantly lower than that in groups B and C ( P<0.05); a small amount of black conductive particles were scattered at the broken end in group D, and the surrounding collagen fibers were less than those in group C. Nissl and LFB staining showed that the structure of neurons and myelin sheath in the injured area of spinal cord in group D was relatively complete and continuous, and the number of Nissl bodies and the positive area of myelin sheath in group D were significantly better than those in groups B and C ( P<0.05). NF200 immunofluorescence staining and Western blot assay results showed that the relative expression of NF200 protein in group D was significantly higher than that in groups B and C ( P<0.05). CD31 immunofluorescence staining showed that the fluorescence intensity of group D was better than that of groups B and C at 28 days after operation, and tubular or linear neovascularization could be seen. The relative expressions of Ang-1 and Tie-2 proteins in group D were significantly higher than those in groups B and C at 7 and 28 days after operation ( P<0.05). The relative expressions of PDGF-B and PDGFR-β proteins in group D were significantly higher than those in groups B and C, and group B was significantly higher than group C at 28 days after operation ( P<0.05). The relative expressions of VEGF-A and VEGFR2 proteins in group D were higher than those in groups B and C, showing significant difference when compared with group B ( P<0.05), but only the expression of VEGF-A protein was significantly higher than that in group C ( P<0.05). There was significant difference only in VEGFR-2 protein between groups B and C ( P<0.05). TGTP may enhance the revascularization of the injured area and protect the neurons, thus alleviating the injury of spinal cord tissue structure and promoting the recovery of neurological function after SCI in rats.

Circ_0036490 and DKK1 competitively bind miR-29a to promote lipopolysaccharides-induced human gingival fibroblasts injury

MicroRNA (miRNA) plays a regulatory role in periodontitis. This study aimed to explore whether miR-29a could affect lipopolysaccharides (LPSs)-induced injury in human gingival fibroblasts (HGFs) through the competitive endogenous RNAs (ceRNA) mechanism. Periodontal ligament (PDL) tissues and HGFs were derived from patients with periodontitis and healthy volunteers. Periodontitis cell model was established by treating HGFs with LPS. Expression levels of circ_0036490, miR-29a, and DKK1 were evaluated by the reverse transcription quantitative real-time PCR (RT-qPCR) method. Western blotting assay was performed to assess protein expression levels of pyroptosis-related proteins and Wnt signalling related proteins. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. Concentration of lactate dehydrogenase (LDH), interleukin (IL)-1β, and IL-18 were determined by Enzyme-linked immunosorbent assay (ELISA). Pyroptosis rate were determined by flow cytometry assay to evaluate pyroptosis. The interaction between miR-29a and circ_0036490 or DKK1 was verified by dual-luciferase reporter and RNA pull-down assays. MiR-29a expression was lower in PDL tissues of patients with periodontitis than that in healthy group; likewise, miR-29a was also downregulated in LPS-treated HGFs. Overexpression of miR-29a increased cell viability and decreased pyroptosis of HGFs induced by LPS while inhibition of miR-29a exerted the opposite role. MiR-29a binds to circ_0036490 and elevation of circ_0036490 contributed to dysfuntion of LPS-treated HGFs and reversed the protection function of elevated miR-29a. In addition, miR-29a targets DKK1. Overexpression of DKK1 abrogated the effects of overexpressed miR-29a on cell vaibility, pyroptosis, and protein levels of Wnt signalling pathway of LPS-treated HGFs. Circ_0036490 and DKK1 competitively bind miR-29a to promote LPS-induced HGF injury in vitro. Wnt pathway inactivated by LPS was activated by miR-29a. Thence, miR-29a may be a promising target for periodontitis.

[Mechanism of resveratrol in protecting ovary in poor ovarian response mice by regulating Hippo signaling pathway].

This study observed the protective effect of resveratrol(Res) on ovarian function in poor ovarian response(POR) mice by regulating the Hippo signaling pathway and explored the potential mechanism of Res in inhibiting ovarian cell apoptosis. Female mice with regular estrous cycles were randomly divided into a blank group, a model group, and low-and high-dose Res groups(20 and 40 mg·kg~(-1)), with 20 mice in each group. The blank group received an equal volume of 0.9% saline solution by gavage, while the model group and Res groups received suspension of glycosides of Triptergium wilfordii(GTW) at 50 mg·kg~(-1) by gavage for two weeks to induce the model. After modeling, the low-and high-dose Res groups were continuously treated with drugs by gavage for two weeks, while the blank group and the model group received an equal volume of 0.9% saline solution by gavage. Ovulation was induced in all groups on the day following the end of treatment. Finally, 12 female mice were randomly selected from each group, and the remaining eight female mice were co-housed with male mice at a ratio of 1∶1. Changes in the estrous cycle of mice were observed using vaginal cytology smears. The number of ovulated eggs, ovarian wet weight, ovarian index, and pregnancy rate of mice were measured. The le-vels of anti-Mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in serum were determined using enzyme-linked immunosorbent assay(ELISA). Ovarian tissue morphology and ovarian cell apoptosis were observed using hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining, respectively. The protein expression levels of yes-associated protein(YAP) 1 and transcriptional coactivator with PDZ-binding motif(TAZ) were detected by immunohistochemistry(IHC), while the changes in protein expression levels of mammalian sterile 20-like kinase(MST) 1/2, large tumor suppressor(LATS) 1/2, YAP1, TAZ, B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were determined by Western blot. The results showed that compared with the blank group, the model group had an increased rate of estrous cycle disruption in mice, a decreased number of normally developing ovarian follicles, an increased number of blocked ovarian follicles, increased ovarian granulosa cell apoptosis, decreased ovulation, reduced ovarian wet weight and ovarian index, increased serum FSH and LH levels, decreased AMH and E_2 levels, decreased protein expression levels of YAP1 and TAZ in ovarian tissues, increased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and decreased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Additionally, the number of embryos per litter significantly decreased after co-housing. Compared with the model group, the low-and high-dose Res groups exhibited reduced estrous cycle disruption rates in mice, varying degrees of improvement in the number and morphology of ovarian follicles, reduced numbers of blocked ovarian follicles, improved ovarian granulosa cell apoptosis, increased ovulation, elevated ovarian wet weight and ovarian index, decreased serum FSH and LH levels, increased AMH and E_2 levels, elevated protein expression levels of YAP1 and TAZ in ovarian tissues, decreased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and increased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Furthermore, the number of embryos per litter increased to varying degrees after co-housing. In conclusion, Res effectively inhibits ovarian cell apoptosis in mice and improves ovarian responsiveness. Its mechanism may be related to the regulation of key molecules in the Hippo pathway.

Pirfenidone and nintedanib attenuate pulmonary fibrosis in mice by inhibiting the expression of JAK2.

Pirfenidone and nintedanib were approved by the Food and Drug Administration (FDA) for the treatment of idiopathic pulmonary fibrosis (IPF). These two drugs can slow the progression of the disease, but the specific mechanisms are not fully understood. In the current study, bleomycin (BLM) induced pulmonary fibrosis in mice was accompanied by high p-JAK2 expression in lung tissue, mainly in the nucleus. The expression of p-JAK2 significantly decreased after intragastric administration of pirfenidone and nintedanib. p-JAK2 is reportedly expressed mainly in the cytoplasm and exerts its effect by activating downstream p-STAT3 in the nucleus. In vivo experiments, pulmonary fibrosis was induced in mice with BLM and then treated with pirfenidone and nintedanib. The levels of transforming growth factor-β (TGF-β1), SP-A, SP-D and KL-6 in serum were measured by enzyme-linked immunosorbent assay (ELISA). Pathological staining was performed to assess lung fibrosis in mice, Western blot was performed to detect the expression levels of relevant proteins, and immunofluorescence was performed to observe the fluorescence expression of p-JAK2. In cellular experiments, MLE12 was stimulated with TGF-β1 and intervened with TGF-β1 receptor inhibitor and si-JAK2, pirfenidone and nintedanib, respectively, and the related protein expression levels were detected by Western blot. In both in vivo and in vitro experiments, pirfenidone and nintedanib were found to attenuate the expression of lung fibrosis markers by inhibiting the expression of JAK2, which may reduce the entry of p-JAK2 into the nucleus by downregulating JAK2 phosphorylation through inhibition of the TGF-β receptor. In contrast, inhibition of JAK2 expression greatly reduced the expression of TGF-β receptor and α-smooth muscles actin (a myofibroblast activation marker). In both in vivo and in vitro experiments, the present study demonstrated that TGF-β1 promotes JAK2 phosphorylation through a non-classical pathway, and conversely, inhibition of JAK2 expression affects the TGF-β1 signalling pathway. Therefore, we speculate that TGF-β1 and JAK2 signaling pathways interact with each other and participate in fibrosis.

Exploring the Osteogenic Potential of Zinc-Doped Magnesium Phosphate Cement (ZMPC): A Novel Material for Orthopedic Bone Defect Repair.

In orthopedics, the repair of bone defects remains challenging. In previous research reports, magnesium phosphate cements (MPCs) were widely used because of their excellent mechanical properties, which have been widely used in the field of orthopedic medicine. We built a new k-struvite (MPC) cement obtained from zinc oxide (ZnO) and assessed its osteogenic properties. Zinc-doped magnesium phosphate cement (ZMPC) is a novel material with good biocompatibility and degradability. This article summarizes the preparation method, physicochemical properties, and biological properties of ZMPC through research on this material. The results show that ZMPC has the same strength and toughness (25.3 ± 1.73 MPa to 20.18 ± 2.11 MPa), that meet the requirements of bone repair. Furthermore, the material can gradually degrade (12.27% ± 1.11% in 28 days) and promote osteogenic differentiation (relative protein expression level increased 2-3 times) of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. In addition, in vivo confirmation revealed increased bone regeneration in a rat calvarial defect model compared with MPC alone. Therefore, ZMPC has broad application prospects and is expected to be an important repair material in the field of orthopedic medicine.

ZNF692 promotes cell proliferation, invasion and migration of human prostate cancer cells by targeting the EMT signaling pathway

BackgroundProstate cancer poses a considerable threat to human health. At present, the mechanism of tumor progression remains unclear. ZNF692 is overexpressed in many tumors, and the high expression of ZNF692 is correlated with tumor aggressiveness and tumor phenotype of prostate cancer, suggesting that ZNF692 may play an important role in tumor biology of prostate cancer. This paper aims to elucidate the relationship between them.MethodsThe expression level of ZNF692 was verified in normal prostate cells (RWPE-1) and prostate cancer cells (LNCaP, PC3, DU145). PC3 cells were selected to construct the ZNF692 knockout prostate cancer cell line. The changes of cell proliferation, apoptosis, invasion and metastasis were detected by CCK8, Edu staining, Transwell assay and scratch assay. The expression levels of related proteins were detected by Western blot.ResultsAt the cellular level, ZNF692 was overexpressed to varying degrees in prostate cancer cell lines, with the highest expression in PC3 cell lines. CCK8 and Edu results showed that the proliferation of prostate cancer PC3 cells that knocked down ZNF692 was slowed. Transwell assay and scratch assay showed reduced invasion and migration of prostate cancer PC3 cells that knocked out ZNF692. Flow cytometry showed that the apoptosis rate of prostate cancer PC3 cells after ZNF692 knockout was increased. In addition, after ZNF692 silencing, the expression level of epithelial phenotype E-cadherin increased in PC3 cells, while the expression level of interstitial phenotype N-cadherin, Vimentin, c-Myc, and CyclinA1 decreased. The state of prostate cancer PC3 cells that overexpressed ZNF692 was reversed from the state after ZNF692 was knocked down.ConclusionZNF692 can be used as a new prognostic marker and a potential biologic therapeutic target for PCa. By inhibiting the expression of c-myc and cyclinA1, the EMT signaling pathway is regulated to provide evidence for its potential molecular mechanism.

Short-term postmortem interval estimation by detection of apoptosis-related protein in skin.

Time-of-death extrapolation has always been one of the most important issues in forensic practice. For a complicated case in which a corpse is destroyed with little evidence, judging the time of death of the deceased is a major challenge, which also enables criminals to escape legal sanctions. To find a method to roughly judge the time of death of a corpse with only a small amount of skin tissue, in this study, we established an early death model by using mice; furthermore, the postmortem interval was estimated by determining the protein and mRNA levels of Bax and Bcl-2 in the skin. In this process, 0h after death was used as the control group, and the expression levels of Bax and Caspase-3 reached the maximum value at 8-12h, while Bcl-2, as an inhibitor of apoptosis protein, peaked after 24h. The mRNA expression levels of related proteins in postmortem skin tissues were also different. The results of these data indicate that the protein and mRNA levels of Bax and Bcl-2 in the skin have potential application in early time-of-death estimation.

FXR overexpression prevents hepatic steatosis through inhibiting AIM2 inflammasome activation in alcoholic liver disease.

Alcoholic liver disease (ALD), a metabolic liver disease caused by excessive alcohol consumption, has attracted increasing attention due to its high prevalence and mortality. Up to date, there is no effective and feasible treatment method for ALD. This study was to investigate whether Farnesoid X receptor (FXR, NR1H4) can alleviate ALD and whether this effect is mediated by inhibiting absent in melanoma 2 (AIM2) inflammasome activation. The difference in FXR expression between normal subjects and ALD patients was analyzed using the Gene Expression Omnibus (GEO) database. Lieber-DeCarli liquid diet with 5% ethanol (v/v) (EtOH) was adopted to establish the mouse ALD model. Liver histopathological changes and the accumulation of lipid droplets were assessed by H&E and Oil Red O staining. Quantitative real-time PCR, Western blotting analysis and immunofluorescence staining were utilized to evaluate the expression levels of related genes and proteins. DCFH-DA staining was adopted to visualize reactive oxidative species (ROS). FXR was distinctly downregulated in liver tissues of patients with steatosis compared to normal livers using the GEO database, and in ethanol-induced AML-12 cellular steatosis model. FXR overexpression ameliorated hepatic lipid metabolism disorder and steatosis induced by ethanol by inhibiting the expression of genes involved in lipid synthesis and inducing the expression of genes responsible for lipid metabolism. Besides, FXR overexpression inhibited ethanol-induced AIM2 inflammasome activation and alleviated oxidative stress and ROS production during ethanol-induced hepatic steatosis. However, when FXR was knocked down, the results were completely opposite. FXR attenuated lipid metabolism disorders and lipid degeneration in alcohol-caused liver injury and alleviated oxidative stress and inflammation by inhibiting AIM2 inflammasome activation.

MiR-26b-5p Affects the Progression of Acute Myeloid Leukemia by Regulating the USP48-Mediated Wnt/β-Catenin Pathway.

Acute myeloid leukemia (AML) is a highly heterogeneous disease. Exploring the pathogenesis of AML is still an important topic in the treatment of AML. The expression levels of miR-26b-5p and USP48 were measured by qRT-PCR. The expression levels of related proteins were detected by Western blot. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Coimmunoprecipitation was used to examine the interaction between USP48 and Wnt5a. Bioinformatics analysis showed that high levels of miR-26b-5p and low levels of USP48 were associated with poor prognosis in AML. miR-26b-5p can negatively regulate the expression of USP48. Downregulation of miR-26b-5p inhibited EMT, cell viability and proliferation of AML cells and accelerated apoptosis. Furthermore, the influence of miR-26b-5p inhibition and USP48 knockdown on AML progression could be reversed by a Wnt/β-catenin signaling pathway inhibitor. This study revealed that miR-26b-5p regulates AML progression, possibly by targeting the USP48-mediated Wnt/β-catenin molecular axis to affect AML cell biological behavior.

Curcumin Modulates NOX Gene Expression and ROS Production via P-Smad3C in TGF-β-Activated Hepatic Stellate Cells.

Liver fibrosis, associated with hepatic stellate cells (HSCs), occurs when a healthy liver sustains damage, thereby impairing its function. NADPH oxidases (NOXs), specifically isoforms 1, 2, and 4, play a role in reactive oxygen species (ROS) production during hepatic injuries, resulting in fibrosis. Curcumin has shown strong potential in mitigating liver fibrosis. Our research aimed to investigate the effects of curcumin on lowering NOX and ROS levels. This compound was also studied for its effects on NOXs, ROS concentrations through the inhibition of Smad3 phosphorylation in transforming growth factor beta (TGF-β)-activated human HSCs. MTT assay investigated the cytotoxic effects of curcumin on HSCs. The cells were activated by exposure to TGF-β (2 ng/mL) for 24 hours. After activating, the cells were treated with curcumin at 25-150 μM concentrations. After administering curcumin to the cells, we employed RT-PCR and Western blot techniques to evaluate the related gene and protein expression levels. This evaluation was primarily focused on the mRNA expression levels of NOX1, NOX2, NOX4 and phosphorylated Smad3C. The mRNA expression level of aforesaid NOXs as well as α-smooth muscle actin (α-SMA), collagen1-α, and ROS levels were significantly reduced following 100 μM curcumin treatment. Furthermore, curcumin significantly decreased the p-Smad3C protein level in TGF-β-activated cells, with fold changes of 3 and 2 observed at 75 and 100 μM, respectively. Curcumin decreased the levels of ROS and NOX, as well as the expression of α-SMA and collagen1-α. The primary mechanism for this reduction could be linked to the level of p-Smad3C. Hence, curcumin could serve as an effective therapeutic agent for liver fibrosis.