Folia histochemica et cytobiologica最新文献

Introduction: Periodontitis (PD) is a chronic inflammatory disease leading to alveolar bone loss. This study investigated the effect of nootkatone and regulatory mechanism in reducing periodontal inflammation and alveolar bone loss in a rat model.

Material and methods: Twenty male Sprague-Dawley rats were divided into control, periodontitis, and nootkatone-treated groups (45 or 90 mg/kg). Ligature induction method was adopted to establish the PD model. After 21 days, rats received daily gavage of either saline or nootkatone for 10 days. Alveolar bone loss was assessed using micro-CT. Histological analyses included hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), and Masson's trichrome stainings. Immunohistochemistry for heme oxygenase 1 (HO-1) and nuclear factor erythroid-2 related factor 2 (Nrf2) were performed in periodontal tissues. Content of inflammatory cytokines IL-1β, IL-6, and TNF-α in gingival tissues around ligature were assessed using ELISA kits. Malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were analyzed and Western blot for NF-κB expression in gingival tissues were performed.

Results: Nootkatone significantly reduced the distance from cementoenamel junction to alveolar bone crest (CEJ-ABC), enhanced bone mineral density (BMD), bone volume (BV), and BV/total volume (TV) ratio in ligature-induced rats. Higher dose of nootkatone (90 mg/kg) did not show more significant therapeutic effect than lower dose (45 mg/kg). Histological staining showed decreased osteoclasts' number and improved bone architecture in the nootkatone group. Content of IL-1β, IL-6, and TNF-α and inflammatory cell infiltration level in gingival tissues around the ligature were decreased in the nootkatone-treatment rats. Nootkatone increased Nrf2 and HO-1 protein expression and decreased NF-κB protein level, suppressing MDA levels and enhancing SOD activity.

Conclusions: In a rat model, nootkatone effectively mitigates periodontal inflammation and alveolar bone loss through the Nrf2/HO-1 and NF-κB pathways. These findings suggest nootkatone as a promising therapeutic agent for the treatment of periodontitis.

Introduction: Pyroptosis is closely related to many chronic diseases including atherosclerosis, but the potential pathomechanisms are still unclear. This study was aimed at exploring how lncRNAs may contribute to pyroptosis and the potential mechanisms.

Material and methods: We performed in vitro assays to investigate the effects of a relatively newly discovered lncRNA, NEXN-AS1, on pyroptosis. Two types of vascular wall cells, i.e. human vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), were used as cell models in this study. A previously constructed lentivirus vector was used to overexpress lncRNA NEXN-AS1 and a small interfering RNA (siRNA) mimic against NEXN to knockdown NEXN. The mRNA and protein levels of molecules of pyroptosis in the canonical inflammasome pathway, including nucleotide-binding oligomerisation segment-like receptor family 3 (NLRP3), caspase-1, gasdermin D (GSDMD), interleukin-1β (IL-1β), and interleukin-18 (IL-18) were detected using quantitative real-time PCR and Western blot analysis, respectively.

Results: We found that lentivirus-mediated overexpression of lncRNA NEXN-AS1 increased the expression levels of NEXN and markedly reduced the expression of critical molecules involved in pyroptosis, including NLRP3, caspase-1, GSDMD, IL-1β, and IL-18, in both VSMCs and ECs. Furthermore, NEXN knockdown could reverse the effects of lncRNA NEXN-AS1 overexpression on pyroptosis.

Conclusions: lncRNA NEXN-AS1 could act as a target for maintaining endothelium homeostasis, increasing plaque stability, and delaying the progression of atherosclerosis.

Introduction: Atopic dermatitis (AD) is the most common allergic skin disease. The dysfunction of keratinocytes is closely associated with AD progression. Nevertheless, the specific functions of CC chemokine ligand 19 (CCL19) and its receptor CC chemokine receptor 7 (CCR7) in human HaCaT keratinocytes are still unclear.

Material and methods: AD cell models in vitro were established by treating HaCaT cells with TNF-alpha (TNF-α, 10 ng/mL) and IFN-gamma (IFN-γ, 10 ng/mL). Cell viability was estimated by MTT assay. The protein levels of CCL19 and CCR7 were tested via Western blotting. The expression of CCL19 protein was knocked down by transfecting si-CCL19 into HaCaT cells. The contents of inflammatory factors i.e. thymus and activation-regulated chemokine (TARC), interleukin 6 (IL-6), and prostaglandin E2 were measured by ELISA, and the nitric oxide content was detected by Griess reagent. The protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were tested via Western blotting.

Results: TNF-α and IFN-γ induced cytotoxicity and upregulated the expression of CCL19 and CCR7 in HaCaT cells. CCL19 knockdown alleviated cytokines-induced cytotoxicity and the release of TARC, IL-6, PGE2 and nitric oxide in TNF-α + IFN-γ-treated HaCaT cells. Furthermore, the protein levels of iNOS and COX-2 were also repressed by CCL19 knockdown. In addition, knockdown of CCL19 decreased CCR7 protein content and inhibited the phosphorylation of IκBα and p65, implying that knockdown of CCL19 inactivated CCR7/NF-κB signalling in HaCaT cells. Rescue assays validated that CCR7 overexpression reversed the effects of CCL19 silencing on the viability and levels of inflammatory factors in TNF-α + IFN-γ-induced HaCaT cells.

Conclusions: This study proves that CCL19 can promote TNF-α + IFN-γ-induced skin inflammatory responses by targeting CCR7/NF-κB pathway in HaCaT cells.

Introduction: Endometriosis (EMs), manifested by pain and infertility, is a chronic inflammatory disease. The precise pathophysiology of this disease remains uncertain. Insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) and polypyrimidine tract-binding protein 1 (PTBP1) have both been found to regulate proliferation, apoptosis, and invasion. This study aimed to investigate the effects of IGF2BP1/PTBP1 in treating EMs.

Materials and methods: qRT-PCR and western blotting were employed to quantify IGF2BP1 and PTBP1 expression in six patients with EMs (mean age 33.83 years). The correlation analysis, STRING database prediction, and RNA immunoprecipitation were utilized to identify the relationship between IGF2BP1 and PTBP1. Ectopic endometrial volume, weight, HE staining, and IGF2BP1 silencing were utilized to estimate the effects of IGF2BP1 in EMs model rats. qRT-PCR, CCK-8, 5-ethynyl-2'-deoxyuridine (EDU) labeling, Transwell assay, and flow cytometry were utilized to assess the effects of IGF2BP1/PTBP1 on the proliferation, migration, invasion, and apoptosis of ectopic endometrial stromal cells (eESCs). Furthermore, western blotting was employed to evaluate expressions of PCNA, VEGF, and E-cadherin in EMs rats and eESCs.

Results: The mRNA and protein levels of IGF2BP1 and PTBP1 in the ectopic and eutopic endometrium of EMs patients were significantly increased. RNA immunoprecipitation revealed a close interaction of IGF2BP1 with PTBP1. Additionally, the endometrial volume, weight, and histopathologic scores in rats were significantly reduced after IGF2BP1 silencing. IGF2BP1 silencing also decreased the expression of PCNA and VEGF, and increased E-cadherin expression in endometrial tissues of EMs rats. Moreover, IGF2BP1 silencing inhibited proliferation, migration, and invasion and promoted apoptosis through PTBP1 in eESCs.

Conclusions: IGF2BP1 exhibits potential beneficial properties in the management of EMs by interacting with PTBP1, thereby highlighting IGF2BP1 as a promising therapeutic target for EMs.

Introduction: During sepsis, the kidney is one of the most vulnerable organs. Sepsis-associated acute kidney injury (S-AKI) is hallmarked by renal inflammation, apoptosis, and oxidative injury. Ginsenoside Rg1 (Rg1) is a natural product that possesses abundant pharmacological actions and protects against many sepsis-related diseases. Nevertheless, its role and related mechanism in S-AKI remain to be determined.

Materials and methods: S-AKI was induced using lipopolysaccharide (LPS, 10 mg/kg) via a single intraperitoneal injection. Rg1 (200 mg/kg) was intraperitoneally administered for 3 consecutive days before LPS treatment. For histopathological examination, murine kidney tissues were stained with hematoxylin and eosin. Tubular injury score was calculated to evaluate kidney injury. Serum creatinine and BUN levels were measured for assessing renal dysfunction. The levels and activities of oxidative stress markers (MDA, 4-HNE, PC, GSH, SOD, and CAT) in renal tissue were measured by corresponding kits. Renal cell apoptosis was detected by TUNEL staining. The protein levels of apoptosis-related markers (Bcl-2, Bax, and Cleaved caspase-3), proinflammatory factors, SIRT1, IκBα, p-NF-κB p65, and NF-κB p65 in kidneys were determined using western blotting. Immunofluorescence staining was employed to assess p-NF-κB p65 expression in renal tissues.

Results: LPS-induced injury of kidneys and renal dysfunction in mice were ameliorated by Rg1. Rg1 also impeded LPS-evoked renal cell apoptosis in kidneys. Moreover, Rg1 attenuated LPS-triggered inflammation and oxidative stress in kidneys by inhibiting proinflammatory cytokine release, enhancing antioxidant levels and activities, and reducing lipid peroxidation. However, all these protective effects of Rg1 in LPS-induced AKI mice were reversed by EX527, an inhibitor of sirtuin 1 (SIRT1). Mechanistically, Rg1 upregulated SIRT1 protein expression, increased SIRT1 activity, and inactivated NF-κB signaling in the kidney of LPS-induced AKI mice, which was also reversed by EX527.

Conclusions: Rg1 ameliorates LPS-induced kidney injury and suppresses renal inflammation, apoptosis, and oxidative stress in mice via regulating the SIRT1/NF-κB signaling.

Introduction: Periodontitis is a serious gum infection that disrupts the soft tissue around teeth. This study aimed to identify the most effective fraction of the Chinese medicine Kangfuxin for periodontitis treatment in a rat model.

Material and methods: Kangfuxin solution was subjected to sequential extraction using chloroform, ethyl acetate, n-butanol, and water. The extracts were evaporated, dissolved in DMSO, diluted in water, and administered to rats via gavage (0.5 mL/day) for 2 weeks. The n-butanol extract was further fractionated using macroporous resin chromatography with 10%, 30%, 50%, 70%, and 90% ethanol elution. Levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in periodontitis samples were examined by ELISA. Leukocyte infiltration in the cementum was analysed by haematoxylin and eosin (H&E) staining.

Results: The n-butanol extract showed the best anti-inflammatory effect, reducing IL-6, IL-1β, and TNF-α levels in periodontitis samples and alleviating tissue damage and leukocyte infiltration in the cementum. Further fractionation revealed that the 50% ethanol fraction of the n-butanol extract had the most potent action in attenuating inflammation. This fraction suppressed the activation of the PI3K-AKT-mTOR signalling pathway in periodontitis samples. Application of a PI3K activator counteracted the anti-inflammatory effect of the 50% ethanol fraction.

Conclusions: We identified a potent anti-inflammatory fraction (50% ethanol fraction of the n-butanol extract) of Kangfuxin for periodontitis treatment. This fraction suppressed the activity of the PI3K-AKT-mTOR signalling pathway in periodontitis samples. Further research is needed to isolate and characterise the specific bioactive compounds within this fraction.

Introduction: Hepatocellular carcinoma (HCC) is the sixth most common type of cancer and the second leading cause of cancer death worldwide [19]. Opioid growth factor (OGF) has been shown to exhibit antitumour potential, binding to OGF receptor (OGFr). Naltrexone (NTX), an OGFr antagonist, is considered as a potential anti-cancer agent. However, the specific mechanism of how OGFr acts on HCC cells is yet to be elucidated.

Materials and methods: HepG2 cells were inoculated into subcutaneous areas of nude mice's back (200 μL, 2.5×10⁷/mL) to establish HCC in vivo models. HepG2 cells were transfected with lentiviral plasmids containing short hairpin RNA (shRNA) targeting OGFr (sh-OGFr) or negative control shRNA (sh-NC), and OGFr over-expression (OE-OGFr) or over-expression negative control (OE-NC) plasmids. Subsequently, male BALB/c nude mice were randomized into Control, sh-NC, sh-OGFr, OE-NC, and OE-OGFr groups (n = 6). Tumour size was measured weekly for four weeks, TUNEL staining for apoptosis, and immunohistochemistry were performed. In vitro, HepG2 cells were randomized into OE-NC, OE-OGFr, and OE-OGFr+NTX (100 μmol/L) groups, and sh-NC, sh-OGFr, sh-OGFr+sh-P21, and sh-OGFr+sh-P16 groups. Cell viability by CCK8 assay, cell proliferation by EDU staining, cell migration by cell scratch, and Western blot were performed.

Results: In vivo, sh-OGFr-transfected HepG2 cells increased tumour weight, volume, and Ki67 expression, decreased P21 and P16 expression, and did not affect apoptosis rate. The effect of OE-OGFr in HepG2 cells was completely the opposite. In vitro, OE-OGFr inhibited HepG2 cells' viability, proliferation, and migration, and further NTX intervention reversed its inhibitory effects. The transfection of HepG2 cells with sh-OGFr+sh-P21 and sh-OGFr+sh-P16 further enhanced the cell proliferation and migration abilities compared to the sh-OGFr group.

Conclusions: OGFr overexpression may inhibit HCC progression by activating P16 and P21 expression to inhibit cell proliferation and migration, thereby providing new potential targets for HCC treatment.

Introduction: Nitric oxide (NO) is present in various cell types in the central nervous system and plays a crucial role in the control of various cellular functions. The diurnal Mongolian gerbil is a member of the rodent family Muridae that exhibits unique physiological, anatomical, and behavioral differences from the nocturnal rat and mouse, which render it a useful model for studying the visual system. The purpose of this study was to confirm the distribution and morphology of neurons that contain nitric oxide synthase (NOS) and their pattern of co-expressing NOS with neuropeptide Y (NPY), somatostatin (SST), and gamma-aminobutyric acid (GABA) in the visual cortex of Mongolian gerbils.

Materials and methods: Mongolian gerbils were used in the study. We confirmed the localization of NOS in the visual cortex of Mongolian gerbils using horseradish peroxidase immunocytochemistry, fluorescent immunocytochemistry, and conventional confocal microscopy.

Results: NOS-immunoreactive (IR) neurons were present in all layers of the visual cortex of the Mongolian gerbil, with the exception of layer I, with the highest density observed in layer V (50.00%). The predominant type of NOS-IR neurons was multipolar round/oval cells (60.96%). Two-color immunofluorescence revealed that 100% NOS-IR neurons were co-labeled with NPY and SST and 34.55% were co-labeled with GABA.

Conclusions: Our findings of the laminar distribution and morphological characteristics of NOS-IR neurons, as well as the colocalization patterns of NOS-IR neurons with NPY, SST, and GABA, indicated the presence of species-specific differences, suggesting the functional diversity of NO in the visual cortex. This study provides valuable data on the anatomical organization of NOS-IR neurons and, consequently, a better understanding of the functional aspects of NO and species diversity.

Introduction: Osteoarthritis (OA) is a prevailing degenerative disease in elderly population and can lead to severe joint dysfunction. Studies have revealed various pharmacological activities of diosmetin, including the anti-OA efficacy. The present study further investigated its effect on interleukin (IL)-1β-induced OA in chondrocytes.

Material and methods: Primary chondrocytes were isolated from young mice, stimulated with IL-1β (10 ng/mL), and pretreated with diosmetin (10 and 20 μM) to conduct the in vitro assays. CCK-8 assay assessed the cytotoxicity of diosmetin whereas the levels of inflammatory factors (PGE2, nitrite, TNF-α, and IL-6) in homogenized cells were evaluated by ELISA. The levels of inflammatory cytokines, content of extracellular matrix (ECM), and signaling-related proteins (Nrf2, HO-1, and NF-κB p65) were assessed by western blotting. Expression of collagen II, p65, and Nrf2 in the chondrocytes was confirmed by immunofluorescence staining. The chondrocytes treated with IL-1β and diosmetin were transfected with Nrf2 knockdown plasmid (si-Nrf2) to investigate the role of Nrf2. In vivo OA mouse model was induced by surgically destabilizing the medial meniscus (DMM). Safranin O staining was conducted to assess the OA severity in the knee-joint tissue.

Results: Diosmetin suppressed the expression of iNOS, COX-2, PGE2, nitrite, TNF-α, IL-6, MMP-13, and ADAMTS-5 induced by IL-1β in chondrocytes. The expression of p-p65, p-IκBα, and nuclear p65 was decreased whereas that of Nrf2 and HO-1 increased by diosmetin treatment in IL-1β-treated chondrocytes. Nrf2 knockdown by siRNA reversed the inhibitory effect of diosmetin on IL-1β-induced degradation of ECM proteins and inflammatory factors in cultured chondrocytes. In the DMM-induced model of OA, diosmetin alleviated cartilage degeneration and decreased the Osteoarthritis Research Society International score.

Conclusions: Diosmetin ameliorates expression of inflammation biomarkers and ECM macromolecules degradation in cultured murine chondrocytes via inactivation of NF-κB signaling by activating Nrf2/HO-1 signaling pathway.

Introduction: Diabetic cataract (DC) is a common ocular complication of diabetes. Mitofusin 2 (MFN2), a mitochondrial fusion protein, is involved in the pathogenesis of cataract and diabetic complications. However, its role and molecular mechanisms in DC remain unclear.

Materials and methods: DC models in rats were induced by intraperitoneal injection of streptozocin (STZ) for 12 weeks. We measured the body weight of rats, blood glucose concentrations, sorbitol dehydrogenase (SDH) activity and advanced glycation end products (AGE) content in the lenses of rats. MFN2 mRNA and protein expression levels in the lenses were detected by RT-qPCR and western blot assays. In vitro, human lens epithelial (HLE) B3 cells were treated for 48 h with 25 mM glucose (high glucose, HG) to induce cell damage. To determine the role of MFN2 in HG-induced cell damage, HLE-B3 cells were transfected with lentivirus loaded with MFN2 overexpression plasmid or short hairpin RNA (shRNA) to overexpress or knock down MFN2 expression, followed by HG exposure. Cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) level. JC-1 staining showed the changes in mitochondrial membrane potential (Δψm). The mediators related to apoptosis, mitochondrial damage, and autophagy were determined.

Results: STZ-administrated rats showed reduced body weight, increased blood glucose levels, elevated SDH activity and AGE content, suggesting successful establishment of the DC rat model. Interestingly, MFN2 expression was significantly downregulated in DC rat lens and HG-induced HLE-B3 cells. Further analysis showed that under HG conditions, MFN2 overexpression enhanced cell viability and inhibited apoptosis accompanied by decreased Bax, cleaved caspase-9 and increased Bcl-2 expression in HLE-B3 cells. MFN2 overexpression also suppressed the mitochondrial damage elicited by HG as manifested by reduced ROS production, recovered Δψm and increased mitochondrial cytochrome c (Cyto c) level. Moreover, MFN2 overexpression increased LC3BⅡ/LC3BⅠ ratio and Beclin-1 expression, but decreased p62 level, and blocked the phosphorylation of mTOR in HG-treated HLE-B3 cells. In contrast, MFN2 silencing exerted opposite effects.

Conclusions: Presented findings indicate that MFN2 expression may be essential for preventing lens epithelial cell apoptosis during development of diabetic cataract.