Proliferating Cell Nuclear Antigen Expression Research Articles

AKTIVITAS ANTITUMOR KOMBINASIEKSTRAKTyphonium flagelliformeDENGANINTERFERON ALAMIANJING DAN KUCING PADA MENCITDENGAN TUMORKULIT YANG DIINDUKSIDMBA

Indonesia has various plants with potential activities and uses as antitumor agents, one of which is Typhonium flagelliforme. This study aimed to examine in vivo the effects of combination therapy of T. flagelliforme leaf-ethanolic extract and natural interferon of canine or feline as an antitumor agent. In this study, 18 female mice of ddY strain induced with 7.12-dimethyl-benz(?)anthracene (DMBA) carcinogenic substance were divided into six treatment groups. All tumor tissues formed were collected and made for histopathological slides. The slides were stained with Hematoxylin-Eosin (HE) and immunohistochemistry (IHC) procedure using antibodies for proliferating cell nuclear antigen (PCNA) and Caspase-3. The macroscopic observations showed that the skin surface of the mice groups with tumors had alopecia with a reddish color. The microscopic observations showed three tumors types: anaplastic carcinoma, fibrosarcoma, and squamous cell carcinoma. The mitotic, angiogenesis, PCNA and Caspase-3 tests showed significant differences among the groups administered with the single and combination test materials. The combination therapy of T. flagelliforme leaf-ethanolic extract and natural interferon can stabilize mice’s body weight, inhibit tumor growth, reduce mitosis and angiogenesis, and reduce PCNA and Caspase-3 expression.

GRK2 Protein Mediates the ANRIL, a lncRNA, to Affect the Proliferation and Apoptosis of Kasumi-1 Cells.

A long non-coding RNAs (LncRNAs) called antisense noncoding RNA in the INK4 locus (ANRIL), has emerged as substantial regulators of cell survival in acute myeloid leukemia (AML). However, its speciffc and potential mechanism is uncertain in AML. In this research, we investigated the role of ANRIL in cell proliferation, apoptosis, and the underlying mechanism in AML cells. ANRIL expression was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Kasumi-1 cells were transfected with LV-ANRIL plasmid to upregulate ANRIL expression, with or without co-transfection with a G Protein-Coupled Receptor Kinase 2 (GRK2) siRNA. Additionally, these cells were transfected with sh-ANRIL plasmid to inhibit ANRIL expression, with or without co-transfection with a GRK2 overexpression plasmid. Cell proliferation and apoptosis were determined using the cell counting kit-8 (CCK8) and flow cytometry. Protein expression levels of phosphatidylinositide 3-kinases (PI3K), protein kinase B (AKT), phosphorylated-Akt (p-AKT), Bcl-2-associated protein x (BAX), B-cell leukemia/lymphoma 2 protein (BCL-2), proliferating cell nuclear antigen (PCNA), cleaved caspase-3, and GRK2 were detected by western blot. The RNA-binding protein immunoprecipitation (RIP) assay was conducted to investigate the interaction between ANRIL and GRK2. ANRIL expression was increased in Kasumi-1 cells. ANRIL upregulation expression promoted cell proliferation and inhibited apoptosis. Furthermore, its upregulation led to increased expressions of PI3K, AKT, p-AKT, PCNA, and BCL-2, and decreased expression of BAX in Kasumi-1 cells. Additionally, transfection with GRK2 siRNA attenuated the promoting effect of LV-ANRIL on Kasumi-1 cells proliferation and the PI3K/AKT pathway, increased BAX and cleaved caspase-3 expressions, and decreased BCL-2 and PCNA expressions. GRK2 overexpression reversed the inhibitory effect of sh-ANRIL on cell proliferation and the PI3K/AKT pathway. Furthermore, it promoted BCL-2 and PCNA expressions, and inhibited BAX and cleaved caspase-3 expressions. RIP assay confirmed the physical interaction between ANRIL and GRK2. The GRK2 protein-mediated ANRIL, increasing Kasumi-1 cell proliferation and inhibiting apoptosis by activating the PI3K/AKT/BCL-2 pathway.

Paricalcitol prevents MAPK pathway activation and inflammation in adriamycin-induced kidney injury in rats.

Activation of the mitogen-activated protein kinase (MAPK) pathway induces uncontrolled cell proliferation in response to inflammatory stimuli. Adriamycin (ADR)-induced nephropathy (ADRN) in rats triggers MAPK activation and pro-inflammatory mechanisms by increasing cytokine secretion, similar to chronic kidney disease (CKD). Activation of the vitamin D receptor (VDR) plays a crucial role in suppressing the expression of inflammatory markers in the kidney and may contribute to reducing cellular proliferation. This study evaluated the effect of pre-treatment with paricalcitol on ADRN in renal inflammation mechanisms. Male Sprague-Dawley rats were implanted with an osmotic minipump containing activated vitamin D (paricalcitol, Zemplar, 6 ng/day) or vehicle (NaCl 0.9%). Two days after implantation, ADR (Fauldoxo, 3.5 mg/kg) or vehicle (NaCl 0.9%) was injected. The rats were divided into four experimental groups: control, n = 6; paricalcitol, n = 6; ADR, n = 7 and, ADR + paricalcitol, n = 7. VDR activation was demonstrated by increased CYP24A1 in renal tissue. Paricalcitol prevented macrophage infiltration in the glomeruli, cortex, and outer medulla, prevented secretion of tumor necrosis factor-α, and interleukin-1β, increased arginase I and decreased arginase II tissue expressions, effects associated with attenuation of MAPK pathways, increased zonula occludens-1, and reduced cell proliferation associated with proliferating cell nuclear antigen expression. Paricalcitol treatment decreased the stromal cell-derived factor 1α/chemokine C-X-C receptor type 4/β-catenin pathway. Paricalcitol plays a renoprotective role by modulating renal inflammation and cell proliferation. These results highlight potential targets for treating CKD.

Telomere transcripts act as tumor suppressor and are associated with favorable prognosis in colorectal cancer with low proliferating cell nuclear antigen expression.

Telomeric repeat-containing RNAs (TERRA) and telomerase RNA component (TERC) regulate telomerase activity (TA) and thereby contribute to telomere homeostasis by influencing telomere length (TL) and the cell immortality hallmark of cancer cells. Additionally, the non-canonical functions of telomerase reverse transcriptase (TERT) and TERRA appear to be involved in the epithelial-mesenchymal transition (EMT), which is important for cancer progression. However, the relationship between TERRA and patient prognosis has not been fully characterized. In this small-scale study, 68 patients with colorectal cancer (CRC) were evaluated for correlations between telomere biology, proliferation, and EMT gene transcripts and disease outcome. The proliferating cell nuclear antigen (PCNA) and the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) showed a positive correlation with TERRA, while TA and TERRA exhibited an inverse correlation. Consistent with previous findings, the present study revealed higher expression levels of TERT and TERC, and increased TA and TL in CRC tumor tissue compared to adjacent non-tumor tissue. In contrast, lower expression levels of TERRA were observed in tumor tissue. Patients with high TERRA expression and low PCNA levels exhibited favorable overall survival rates compared to individuals with the inverse pattern. Furthermore, TERRA suppressed CRC tumor growth in severe combined immunodeficiency disease (SCID) mice. In conclusion, our study extends previously published research on TERRA suggesting its potential therapeutic role in telomerase-positive CRC.

Comparison of Proliferating Cell Nuclear Antigen (PCNA) Expression among Nasal Polyp and Chronic Rhinosinusitis

Background and objectiveChronic rhinosinusitis is a common inflammatory disorder in sinonasal mucosa that could be developed with or without nasal polyps. Cellular proliferation is suggested as a possible mechanism of nasal polyp development. However, conducted studies in this context are limited. So, the present study's aim is the comparison of proliferating cell nuclear antigen (PCNA) expression in nasal polyps and chronic rhinosinusitis. Materials and methodsIn this cross-sectional study, 70 nasal polyp and 60 chronic rhinosinusitis samples from patients referred to Mostafa Khomeini Hospital, Tehran from 2017 to 2022 were immunohistochemically stained by PCNA marker. The percentage of PCNA nuclear expression was determined in two groups and its association with the type of pathological lesion and the patient's age and sex was analyzed by SPSS statistic software version 24 statistical software (IBM Statistics, USA). ResultsThe mean percentage expression of PCNA in nasal polyp and chronic rhinosinusitis samples was 16.55 ± 13.66 and 17.58 ± 12.68 respectively (ranging from 0 to 57 in both groups) however, there was no significant statistical difference between the two groups (p = 0.479). No relationship was found between PCNA expression with age and sex in none of the chronic rhinosinusitis and nasal polyp groups. ConclusionProliferative activity of the nasal epithelial cell is similar in chronic rhinosinusitis with and without nasal polyps and it is considered that the increase of epithelial cell proliferative activity probably has no role in nasal polyp development in patients with chronic rhinosinusitis.

Diterpene glycosides from Fructus Rubi ameliorates benign prostatic hyperplasia in rats through the androgen and TGF-β/Smad signaling pathway

Fructus Rubi (FR), a food material with medicinal value, is used in traditional Chinese medicine (TCM) for treatment of various kidney-related problems, such as impotence, spermatorrhea, and frequent urination. It is also frequently used to produce diverse functional foods in China. The purpose of this research was to assess the therapeutic effects of FR diterpene glycosides on RWPE-1 epithelial cell (RWPE-1), a human normal prostatic epithelial cell, and benign prostate hyperplasia (BPH) rats, both of which had been exposed to dihydrotestosterone (DHT) and testosterone propionate (TP), respectively, and to investigate the mechanism of action. Target proteins that could stably bind to certain diterpene glycosides were screened through drug affinity responsive target stability combined with mass spectrometry (DARTS/MS). DHT-induced RWPE-1cells were used to detect drug activity. TP was subcutaneously injected to induce BPH in rats. The extract of diterpene glycosides from FR (FDS) was orally administered for 28 days. The DHT levels in the serum and prostate tissue of the rats were measured through enzyme-linked immunosorbent assay (ELISA), and to analyze cell proliferation and epithelial-mesenchymal transition (EMT), the protein expression of prostate-specific antigen (PSA), androgen receptor (AR), steroid 5α-reductase type 2 (SRD5A2), proliferating cell nuclear antigen (PCNA), S100 calcium-binding protein A2 (S100A2), transforming growth factor-β1 (TGF-β1), E-cadherin, vimentin, and Smad4 was determined through western blotting (WB), immunohistochemistry (IHC), or immunofluorescence (IF). FDS reduced the proliferation of DHT-induced RWPE-1cells. It also significantly inhibited rat prostate enlargement; decreased DHT levels in the serum and prostate tissue; inhibited the protein expression of AR, PSA, PCNA, S100A2, TGF-β1, E-cadherin, and Smad4; and increased the protein expression of E-cadherin. The present study is the first to report that diterpene glycosides isolated from FR inhibited BPH at the cellular level, regulated the proliferation of prostate cells through the androgen signaling pathway, and prevented EMT in the prostate through the S100A2-mediated TGF-β/Smad signaling pathway. These results indicate that FDS is a promising multitarget therapy for BPH.

Role of hsa_circ_0007482 in pterygium development: insights into proliferation, apoptosis, and clinical correlations.

To investigate the impact of hsa_circ_0007482 on the proliferation and apoptosis of human pterygium fibroblasts (HPFs) and its correlation with the severity grades of pterygium. Pterygium and normal conjunctival tissues were collected from the superior area of the same patient's eye (n=33). The correlation between pterygium severity and hsa_circ_0007482 expression using quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were analyzed. Three distinct siRNA sequences targeting hsa_circ_0007482, along with a negative control sequence, were transfected into HPFs. Cell proliferation was assessed using the cell counting kit-8. Expression levels of Ki67, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bax, B-cell lymphoma-2 (Bcl-2), and Caspase-3 were measured via RT-qPCR. Immunofluorescence staining was employed to detect Ki67 and vimentin expressions. Apoptosis was evaluated using flow cytometry. Hsa_circ_0007482 expression was significantly higher in pterygium tissues compared to normal conjunctival tissues (P<0.001). Positive correlations were observed between hsa_circ_0007482 expression and pterygium severity, thickness, and vascular density. Knockdown of hsa_circ_0007482 inhibited cell proliferation, reducing the mRNA expression of Ki67, PCNA, and Cyclin D1 in HPFs. Hsa_circ_0007482 knockdown induced apoptosis, increasing mRNA expression levels of Bax and Caspase-3, while decreasing Bcl-2 expression in HPFs. Additionally, hsa_circ_0007482 knockdown attenuated vimentin expression in HPFs. The downregulation of hsa_circ_0007482 effectively hampers cell proliferation and triggers apoptosis in HPFs. There are discernible positive correlations detected between the expression of hsa_circ_0007482 and the severity of pterygium.

Ameliorating and Therapeutic Impact of Curcumin Nanoparticles Against Aluminum Oxide Nanoparticles Induced Kidney Toxicity, DNA Damage, Oxidative Stress, PCNA and TNFα Alteration in Male Rats.

Aluminum oxide nanoparticles (Al2O3 NPs) are among the most extensively utilized nanoparticles in nanotechnology and that have negative impacts on the environment. Therefore, the intention of this work is to investigate the protective and therapeutic effects of curcumin in nanoform (Cur NPs) against Al2O3 NPs induced kidney toxicity, oxidative stress, DNA damage, and changes in necrosis factor alpha (TNFα) and proliferating cell nuclear antigen (PCNA) expressions in male rats. Fifty healthy adult male were divided into five groups [G1, control; G2, received 50 mg/kg/day for 4 weeks of Cur NPs orally; G3, received 6 mg/kg BW orally for 4 weeks of Al2O3 NPs; G4, (Cur NPs + Al2O3 NPs) received Cur NPs and Al2O3 NPs at a dose similar to G2 and G3, respectively for 4 weeks; G5, (Al2O3 NPs + Cur NPs) received Al2O3 NPs at a dose similar to G3 for 4 weeks then received Cur NPs at a dose similar to G2 for another 4 weeks]. Current results revealed that Al2O3 NPs induced a significant elevation in serum urea, creatinine, chloride, calcium, kidney malondialdehyde (MDA), DNA damage, injury, TNFα and PCNA expressions and a significant depletion in serum potassium, kidney superoxide dismutase (SOD), glutathione (GSH) as compared to control. On the other hand, treatments of Al2O3 NPs with Cur NPs induced modulation in all altered parameters and improved kidney functions and structure, with best results for the Al2O3 NPs + Cur NPs than Cur NPs + Al2O3 NPs. In conclusion, Cur NPs has the capacity to mitigate the renal toxicity induced by Al2O3 NPs in male albino rats.

Does Varying Platelet-Rich Fibrin Centri̇fugati̇on Protocols Enhance New Bone Formati̇on in Extracti̇on Site?

Finding a protocol that could prevent bone resorption and be implemented in clinical practice would be crucial in providing sufficient bone to replace missing teeth with implants. The study aimed to determine the effectiveness of different centrifugation platelet-rich fibrin (PRF) protocols in new bone formation and bone regenerative markers. This randomized clinical trial was conducted at Izmir Katip Çelebi Research Hospital, a population-based facility in Izmir. Study subjects were composed of patients who required extraction of anterior teeth. Exclusion criteria included periodontal disease, resorption of alveolar bone, defects, smoking, alcoholism, and systemic diseases. The independent variable was the PRF protocol. The subjects were randomly assigned to one of three groups: Leukocyte Platelet-Rich Fibrin (L-PRF), Advanced Platelet-Rich Fibrin (A-PRF) and control groups (healing naturally). The primary outcome of interest was the percentage of new bone formation, determined by analyzing the staining intensity in histomorphometric assessments of bone samples collected 8weeks after extraction. The secondary outcomes were regenerative effects measured by the immunohistochemical expression of markers such as osteocalcin, alkaline phosphatase, and proliferating cell nuclear antigen. Potential benefits were evaluated by clinical observations of pain, swelling, membrane visibility and healing. Age, sex and health conditions. Histologic comparative staining intensities and biomarkers expression between groups were evaluated by one way analysis of variance. A difference of P<.05 was considered statistically significant. The study included 57 subjects, with a mean age of 45years (±5.6); 29 were male (51%) and 28 female (49%). The control group had a mean new bone formation of 32.68% (±2.5), the A-PRF group 61.37% (±3.0), and the L-PRF group 70.74% (±3.5) (P<.001). The A-PRF group showed significantly higher osteocalcin expression than the control group (P=.013). Alkaline phosphatase and proliferating cell nuclear antigen expression scores for PRF groups were significantly higher than the control group's (P=.001). Both groups demonstrated significantly lower pain scores, reduced gingival swelling, better membrane visibility, and healing compared to the control group. PRF enhanced bone formation rates, with L-PRF showing the most significant effect.

Mechanism of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata herbal pair in promoting hepatocellular regeneration in chronic acute liver failure based on HGF/PI3K/Akt signaling axis

Based on the hepatocyte growth factor(HGF)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling axis, this study investigated the therapeutic effect of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata(PRR-ALRP) her-bal pair on acute-on-chronic liver failure(ACLF) rats and its impact on hepatocellular regeneration. The rat model of ACLF was constructed by subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of lipopolysaccharides(LPS)+D-galactosamine(D-GalN). The rats were divided into normal control(NC) group, model(vehicle) group, PRR-ALRP(5.85 g·kg~(-1)) group, and hepatocyte growth factor granules(HGFG, 4.05 g·kg~(-1)) group. Hematoxylin-eosin(HE) staining was used to observe pathological changes in rat liver tissues. Serum alanine aminotransferase(ALT), aspartate transaminase(AST), and total bilirubin(TBIL) were detected using an automatic biochemical analyzer. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) inflammatory factors were detected by ELISA. Immunofluorescence staining was used to detect the positive expression of proliferating cell nuclear antigen(PCNA), antigen identified by monoclonal antibody(Ki67), and cell cycle protein B1(CyclinB1). Real-time fluorescence-based quantitative polymerase chain reaction(RT-qPCR) and Western blot were used to detect the mRNA and protein expression levels of HGF, growth factor receptor-bound protein 1(Gab1), PI3K, Akt, phosphorylated PI3K(p-PI3K), and phosphorylated Akt(p-Akt). The results showed that compared with the vehicle group, the PRR-ALRP group had reduced liver tissue pathological scores, improved liver function, and reduced inflammatory response, with enhanced PCNA, Ki67, and CyclinB1 fluorescence expression. Furthermore, compared with the model group, the PRR-ALRP group showed upregulated expression of HGF and Gab1 proteins, as well as activation of PI3K and Akt phosphorylation. These findings suggest that PRR-ALRP herbal pair exerts anti-liver failure effects by alleviating hepatocyte inflammatory damage and promoting hepatocellular regeneration, and its specific regulatory mechanism may be related to the activation of the HGF/PI3K/Akt signaling pathway.

Modulation of the intestinal mucosal and cell-mediated response against natural helminth infection in the African catfish Clarias gariepinus

Fish gut is a versatile organ serving as the primary pathway for invasion by pathogens, particularly parasites, playing a crucial role in modulating the intestinal adaptive immune response. This study aimed to investigate the cellular-mediated reaction, mucosal acidity, and the expression of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), and CD68 in the intestines of catfish, Clarias gariepinus, naturally infected with helminths. Forty catfish were collected from the Nile River and examined for intestinal parasites. The intestinal tissues of the control and infected fish were fixed for histochemical and immunohistochemical studies. Two groups of helminths were found: cestodes Tetracampos ciliotheca and Polyonchobothrium clarias, and nematodes Paracamallanus cyathopharynx, with a prevalence rate of 63.63%, 18.0%, and 18.0%, respectively. Our results showed that the infected fish had a statistically significant rise in the activity of immune cells, including mast cells, eosinophil granular cells, and dendritic cells. This correlated with upregulation in the expressions of PCNA, VEGF, and CD68. Histochemical analyses demonstrated a marked increase in acidic mucus production, Sudan black B, and bromophenol mercury blue. This study enriches our understanding of the evolution of vertebrate immunity in combating intestinal parasitic infections and the host’s adaptive responses.

Protective effects of 2-methoxyestradiol against hypoxic pulmonary hypertension in neonatal rats

To investigate the protective effects of 2-methoxyestradiol (2ME) against hypoxic pulmonary hypertension (HPH) in neonatal rats. Ninety-six Wistar neonatal rats were randomly divided into a normoxia group, a hypoxia group, and a hypoxia + 2ME group, with each group further subdivided into 3-day, 7-day, 14-day, and 21-day subgroups, containing eight rats each. The hypoxia and hypoxia + 2ME groups received daily subcutaneous injections of saline and 2ME (240 μg/kg), respectively, while the normoxia group was raised in a normoxic environment with daily saline injections. Right ventricular systolic pressure (RVSP) was measured using the direct pressure method. Pulmonary vascular morphology was assessed using hematoxylin and eosin staining, with metrics including the percentage of medial thickness of small pulmonary arteries relative to the external diameter (MT%) and the cross-sectional area of the media of small pulmonary arteries relative to the total cross-sectional area (MA%). Immunohistochemistry was used to detect the expression levels of hypoxia-inducible factor-1α (HIF-1α) and proliferating cell nuclear antigen (PCNA) proteins, while real-time quantitative PCR was used to to assess HIF-1α and PCNA mRNA levels. Compared to the normoxia group, the hypoxia and hypoxia + 2ME groups showed increased RVSP and upregulated HIF-1α and PCNA protein and mRNA expression levels at 3, 7, 14, and 21 days after hypoxia (P<0.05). Furthermore, at 7, 14, and 21 days after hypoxia, the hypoxia group showed increased MT% and MA% (P<0.05). In comparison to the hypoxia group, the hypoxia + 2ME group exhibited reduced RVSP and downregulated HIF-1α and PCNA protein and mRNA expression levels, along with decreased MT% and MA% at 7, 14, and 21 days after hypoxia (P<0.05). 2ME may protect against HPH in neonatal rats by inhibiting the expression of HIF-1α and PCNA and reducing pulmonary vascular remodeling. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 757-764.

Revealing New Prospects: Antipsychotic Drugs Exert Anti-Tumor Effects against Gastric Cancer through Inducing Apoptosis.

Globally, Gastric Cancer (GC) ranks as the fifth leading cause of cancer-related deaths. GC is a multifaceted malignancy with diverse etiologies; however, understanding the shared molecular mechanisms can aid in discovering novel targeted therapies for GC. This study has employed a drug repositioning approach to explore new drug candidates for treating GC. The human GC cell lines AGS, MKN-45, and KATO-III were treated with different concentrations of dopamine, cabergoline, thioridazine, and entacapone to determine effective doses and IC50 values. In vitro, cytotoxic activity on cancer cell lines was screened based on dose/time using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) was used to measure the mRNA expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Proliferating Cell Nuclear Antigen (PCNA) in each group. The percentage of apoptotic cells was evaluated using Annexin V/PI staining. Dopamine, cabergoline, thioridazine, and entacapone elicited cytotoxic effects on AGS and KATO-III cells in a dose-dependent manner and elevated the percentage of Annexin V-positive cells, suggesting the occurrence of apoptosis. The expression of Bcl-2 and PCNA was significantly decreased, whereas the expression of Bax was considerably increased in the AGS and KATO-III cells compared to that in the blank group (p < 0.05); however, no similar effect was observed in MKN-45 cells. Through in vitro experiments, this study provides evidence that the antipsychotic drugs cabergoline, dopamine, thioridazine, and entacapone can inhibit gastric cancer growth in AGS and KATO-III cells. These findings suggest that these drugs could be repurposed as novel therapeutic agents for the treatment of gastric cancer.

Multiple-heated cooking oil promotes early hepatic and renal senescence in adult male rats: the potential regenerative capacity of oleuropein

For economic purposes, cooking oil is repeatedly heated in food preparation, which imposes serious health threats. This study investigated the detrimental effects of multiple-heated cooking oil (MHO) on hepatic and renal tissues with particular focusing on cellular senescence (CS), and the potential regenerative capacity of oleuropein (OLE). Adult male rats were fed MHO-enriched diet for 8 weeks and OLE (50 mg/kg, PO) was administered daily for the last four weeks. Liver and kidney functions and oxidative stress markers were measured. Cell cycle markers p53, p21, cyclin D, and proliferating cell nuclear antigen (PCNA) were evaluated in hepatic and renal tissues. Tumor necrosis factor-α (TNF-α) and Bax were assessed by immunohistochemistry. General histology and collagen deposition were also examined. MHO disturbed hepatic and renal structures and functions. MHO-fed rats showed increased oxidative stress, TNF-α, Bax, and fibrosis in liver and kidney tissues. MHO also enhanced the renal and hepatic expression of p53, p21, cyclin D and PCNA. On the contrary, OLE mitigated MHO-induced oxidative stress, inflammatory burden, apoptotic and fibrotic changes. OLE also suppressed CS and preserved kidney and liver functions. Collectively, OLE displays marked regenerative capacity against MHO-induced hepatic and renal CS, via its potent antioxidant and anti-inflammatory effects.

Histopathological and Immunohistochemical Studies of a Spontaneous Case of Pulmonary Adenomatosis in an Adult Ram: A Case Report

Aim: The present study aimed to describe histopathological findings and immunohistochemical expression of proliferating cell nuclear antigen (PCNA) in a spontaneous case of pulmonary adenomatosis in an adult ram. Case Presentation: A carcass of an adult ram was presented for necropsy in the Post-mortem Hall of the Department of Veterinary Pathology, College of Veterinary Sciences of the Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar with a history of respiratory distress. A detailed necropsy was performed. The tracheal mucosa showed mild congestion. The lungs were pale and firm in consistency. On histopathological examination, nasal turbinate and trachea both showed the vascular changes along with mild infiltration of mononuclear cells. The sections of lungs revealed pulmonary adenocarcinoma characterized by the presence of cuboidal to low columnar epithelial cells forming acinar or papillary-like structures in the parenchyma and surrounded by the normal alveoli. Areas of necrosis along with infiltration of mononuclear cells were also evident. An Immunohistochemical study of the lung sections revealed mild to moderate immunopositive reactivity for the PCNA in the nuclei of neoplastic cells of adenocarcinoma. It indicated the proliferative activity of neoplastic cells. Conclusion: Based on histopathological examination, the present case was diagnosed as pulmonary adenomatosis. PCNA expression indicated the presence of actively proliferating neoplastic cells.

Plasma Proteomics Identifies B2M as a Regulator of Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction.

Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear. We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples. Plasma proteomics identified high protein abundance levels of B2M (β2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF. Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

Citropten Inhibits Vascular Smooth Muscle Cell Proliferation and Migration via the TRPV1 Receptor.

Vascular smooth muscle cell (VSMC) proliferation and migration play critical roles in arterial remodeling. Citropten, a natural organic compound belonging to coumarin and its derivative classes, exhibits various biological activities. However, mechanisms by which citropten protects against vascular remodeling remain unknown. Therefore, in this study, we investigated the inhibitory effects of citropten on VSMC proliferation and migration under high-glucose (HG) stimulation. Citropten abolished the proliferation and migration of rat vascular smooth muscle cells (RVSMCs) in a concentration-dependent manner. Also, citropten inhibited the expression of proliferation-related proteins, including proliferating cell nuclear antigen (PCNA), cyclin E1, cyclin D1, and migration-related markers such as matrix metalloproteinase (MMP), MMP2 and MMP9, in a concentration-dependent manner. In addition, citropten inhibited the phosphorylation of ERK and AKT, as well as hypoxia-inducible factor-1α (HIF-1α) expression, mediated to the Krüppel-like factor 4 (KLF4) transcription factor. Using pharmacological inhibitors of ERK, AKT, and HIF-1α also strongly blocked the expression of MMP9, PCNA, and cyclin D1, as well as migration and the proliferation rate. Finally, molecular docking suggested that citropten docked onto the binding site of transient receptor potential vanilloid 1 (TRPV1), like epigallocatechin gallate (EGCG), a well-known agonist of TRPV1. These data suggest that citropten inhibits VSMC proliferation and migration by activating the TRPV1 channel.

Anti-proliferative effect and mechanisms of Peony pollen on BPH via inhibition of inflammatory factors, oxidative damage and modulation of gut microbiota and SCFAs metabolism

BackgroundPeony pollen (PP) has long been used as a functional food and herbal medicine. However, the potential protective effect and mechanisms of PP on benign prostatic hyperplasia (BPH) are still unclear. This study aims to explore the therapeutic effect and potential mechanisms of PP on BPH. MethodsThe rat model of BPH was induced by subcutaneous injection of testosterone propionate (TP). Meanwhile, the model rats were treated with PP, and physiological and biochemical indexes, the steroid 5 alpha reductase 2 (SRD5A2), proliferating cell nuclear antigen (PCNA) and androgen receptor (AR) expressions, gut microbiota and short-chain fatty acids (SCFAs) were measured. ResultsThe results showed that the wet weight of prostate tissue and the thickness of epithelium decreased treated with PP significantly. In addition, the PP also adjusted the androgen levels, down-regulated the SRD5A2, PCNA and AR expressions, inhibited the expression of inflammatory factors including interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and reduced oxidative damage in BPH rats. It is worth noting that PP can obviously regulate the gut microbiota disorder of BPH rats, increase the abundance of beneficial bacteria (e.g., Romboutsia), reduce the abundance of pathogenic bacteria (e.g., norank_f_Oscillospiraceae), and stimulate the increase of SCFAs content. ConclusionsThese data showed that PP can resist abnormal proliferation of the prostate by down regulation of SRD5A2, PCNA and AR expressions, attenuating oxidation stress and inflammatory damages and directly regulate the gut microbiota, especially the Firmicutes, and increased the level of SCFAs to ameliorate BPH. Therefore, PP might be an excellent natural plant for the development of BPH-related drugs or dietary supplements.

Hinokiol regulates the cell cycle and apoptosis of nasopharyngeal carcinoma CNE1 cells via Hippo-YAP signaling pathway

Objective: To explore the effects of hinokiol on the cell cyle and apoptosis of CNE1 nasopharyngeal carcinoma cells and the relevant molecular mechanism. Methods: The CNE1 cells were cultured in vitro and incubated with different concentrations of honokiol, and the cells were divided into blank control group, 10 μmol/L, 20 μmol/L and 40 μmol/L hinokiol treatment groups, and 10 μg/ml cisplatin group. Cell viability was determined by methylthiazolyldiphenyl- tetrazolium bromide (MTT) method, the cell cycle distribution was detected by flow cytometry, mitochondrial membrane potential was detected by mitochondrial membrane potential test kit, apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method, and the proteins expression of proliferating cell nuclear antigen (PCNA) and G1/S specific cyclin D1 (cyclin D1) were detected by immunoblotting. RNA-Seq was conducted in the hinokiol-treated cells. The mRNA expression of yes-associated protein delta (YAP) was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The proteins expression of phosphor-YAP (p-YAP) and nuclear YAP were detected by immunoblotting, the nuclear distribution of YAP protein was detected by immunofluorescence in the cells with or without treated with the mammalian STE20-like kinase 1/2 (MST1/2) inhibitor (XMU-MP-1), hinokiol, and XMU-MP-1+hinokiol. Statistical analysis of the data was conducted using GraphPad Prism 8.0 software. Resluts Compared with the control group, the cell viablity of CNE1 cells, the levels of mitochondrial membrane potential, the proteins expression of PCNA and cyclin D1 in hinokiol treatment groups were markedly decreased (all P values<0.05), while the proportion of G0/G1 phase cells and the ratio of TUNEL-positive cells were significantly increased (both P values<0.05). Transcriptome analysis showed that differential genes were mainly enriched in Wnt signaling pathway, tumor necrosis factor pathway, and Hippo signaling pathway. The mRNA level of YAP and the protein expression of YAP in the nucleus were decreased and the level of p-YAP protein was increased in cells treated with hinokiol, which were significantly different from control group (all P values<0.05). Compared with the hinokiol group, XMU-MP-1+hinokiol groups showed the decrease of p-YAP protein expression (1.157±0.076 vs 0.479±0.038, t=37.120, P<0.05), the increase of YAP protein expression in the nucleus (0.143±0.012 vs 0.425±0.031, t=29.181, P<0.05), the reduced proportion of cells in G0/G1 phase [(72.494±3.309)% vs (58.747±2.865)%, t=17.265, P<0.05], and the decrease of apoptosis ratio [(53.158±3.376)% vs (29.621±2.713)%, t=28.584, P<0.05]. Conclusion: Hinokiol can arrest the cell cycle and induce the cell apoptosis of CNE1 cells via Hippo/YAP signaling pathway.

Rapid and simple isolation of precartilaginous stem cells: A novel approach without immunomagnetic bead sorting

ObjectiveThe present investigation was designed to devise a rapid and straightforward technique for the isolation of rat precartilaginous stem cells (PCSCs) that eschews the use of immunomagnetic bead sorting. MethodRat neonates within 24 h of birth were selected for this study. Microsurgical techniques were used to harvest the femur, tibia, and the musculature of the knee joint. The ring of LaCroix between the metaphysis of the femur and the epiphysis was excised and divided into fragments of approximately 1 mm³. Tissue sections were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 medium supplemented with 20 % fetal bovine serum and 1 % penicillin-streptomycin. Cell digestion and passaging were performed using trypsin when cells reached 70%–80 % confluence. Third-generation cells underwent immunofluorescence staining and flow cytometry to evaluate fibroblast growth factor receptor-3(FGFR-3) and proliferating cell nuclear antigen(PCNA) expression, while β-galactosidase staining was used to determine cellular senescence. ResultsWithin two days of isolation, numerous short spindle-shaped cells exhibiting distinct refractive properties were observed around the tissue fragments. These cells began to proliferate within 2–3 days and displayed ample cytoplasm. Adherent cells adopted various morphologies, including angular, triangular, and elongated spindles. By the fifth day, more than 80 % of the culture dish surface was covered with elongated cells, with some arranged in patterns reminiscent of whirlpools. Significant FGFR-3 and PCNA expression was confirmed via immunofluorescence in the third-generation cells. Additionally, flow cytometry identified that the proportion of cells positive for FGFR-3 and PCNA exceeded 98 %. Notably, the cells preserved their proliferative capacity through nine passages in vitro, with a marginal proportion showing senescence as indicated by β-galactosidase staining alone. ConclusionThe developed tissue adherence protocol was used to successfully isolate PCSCs with positive FGFR-3 and PCNA expression, rendering the immunomagnetic bead sorting superfluous. The expression of FGFR-3 and PCNA in the isolated cells persisted through the ninth passage in vitro with minimal senescence.