Characteristics and reference ranges of CD4+T cell subpopulations among healthy adult Han Chinese in Shanxi Province, North China.

BACKGROUND: Immunophenotyping of blood lymphocytes is an essential tool to evaluate the immune function of patients with immunodeficiency or autoimmunity. Predominately identified CD4+T cell subsets, Th1, Th2, Th17, as well as regulatory T (Treg) cells, play crucial roles in several immunological and pathological conditions. Considering the variations in cell counts among populations and ethnicities, specific CD4+T cell subset reference values need to be locally established to enable meaningful comparisons and accurate data interpretation in clinical and research settings. Therefore, the aim of this study was to establish distributions and reference ranges for blood CD4+T cell subpopulations in age- and sex-balanced healthy adults of a Han Chinese population in Shanxi Province, North China. METHODS: Peripheral blood CD4+T cell subsets were examined in 150 healthy volunteers (75 males, 75 females) aged 20-70 years with a four-color FACSCalibur flow cytometer. RESULTS: Reference value percentages (absolute counts, cells/µl) were defined as 95% of the population for cell types as follows: CD4+T, 23.78-51.07 (360-1127); Th1, 0.43-39.62 (2.64-276.21); Th2, 0.27-3.57 (1.80-27.14); Th17, 0.22-2.62 (1.10-19.54); and Treg, 2.17-7.94 (13.47-64.58). The ranges for the Th1:Th2 and Th17:Treg ratios were 0.59-52.37 and 0.04-0.76, respectively. Notably, a significant increase was observed in the values of Treg cells in older individuals, and the numbers of Treg cells in females also tended to decrease when compared to those in males. Therefore, we established the distribution and reference range of CD4+T cell subsets based on age and sex, demonstrating the lowest values of Treg cells in younger females. CONCLUSIONS: Collectively, our data provide population-, age-, and sex-specific distributions and reference ranges of circulating CD4+T cell subpopulations, which can be adopted to guide clinical decisions and interpretation of immunophenotyping data in the Han Chinese population in Taiyuan, Shanxi Province, China. In addition, the low expression of peripheral Treg cells in younger females may be associated with the predisposition of females to autoimmune diseases.

Sirolimus selectively increases circulating Treg cell numbers and restores the Th17/Treg balance in rheumatoid arthritis patients with low disease activity or in DAS28 remission who previously received conventional disease-modifying anti-rheumatic drugs.

OBJECTIVES: Regulatory T (Treg) cells are crucial players in the prevention of autoimmunity. Mechanistic target of rapamycin (mTOR) signalling negatively controls the development and function of Treg cells. The aim of the present study was to evaluate the effects of rapamycin, under the generic name sirolimus, on CD4+CD25+FoxP3+ Treg cells in rheumatoid arthritis (RA) patients with low disease activity or in DAS28 remission. METHODS: Fifty-five RA patients and 60 healthy controls were enrolled in this study. All patients had previously received conventional disease-modifying anti-rheumatic drugs (DMARDs) and were considered to have a low DAS28 score (≤3.2). Peripheral blood samples and clinical information were obtained at baseline and following 6 and 12 weeks of sirolimus treatment, or after 12 weeks of conventional treatment. Peripheral blood samples were also obtained from the healthy controls. The circulating levels of lymphocyte subpopulations were assessed by flow cytometry. RESULTS: Thirty-five patients received sirolimus and 20 patients continued treatment with conventional DMARDs. The absolute counts and proportions of CD4+CD25+FoxP3+ Treg cells were significantly lower in all RA patients with DAS28 ≤ 3.2 as compared with those in healthy controls. By contrast, the difference in circulating Th17 cell numbers was not significant. Sirolimus administration resulted in elevations in circulating Treg cell numbers and significant reductions in the Th17/Treg cell ratio, whereas the circulating level of Treg cells and the Th17/Treg cell ratio in patients under conventional treatment both showed a tendency of reduction. Furthermore, a greater proportion of patients under sirolimus treatment achieved DAS28-based remission at 12 weeks. CONCLUSIONS: Sirolimus can favourably expand Treg cells in RA patients with DAS28 ≤3.2, consequently restoring a healthy balance of Th17/Treg cells, which might improve the likelihood of long-term and sustained clinical remission and reduce the probability of disease flare-ups in RA.

Induction of pathological changes and impaired expression of cytokines in developing female rat spleen after chronic excess fluoride exposure.

This study was designed to investigate the effects of excessive fluoride on spleen toxicity. Twenty-four healthy female rats were randomly divided into two groups, each of 12 rats. Each group of female rats was given a control diet and either F- = 0 mg/L or an excessive F- = 150 mg/L in the drinking water for 120 days. The histomorphological and ultrastructural changes in their splenic tissues were observed under light and transmission electron microscopes. DNA damage and splenocyte apoptosis were examined using the micronucleus (MN) assay, single-cell gel electrophoresis (SCGE), and flow cytometry. The expression levels of cytokines, including interleukin (IL)-1ß, IL-2, IL-6, and tumor necrosis factor (TNF)-α, were determined through immunohistochemistry and Western-blot analysis. Results demonstrated that the histomorphological characteristics and ultrastructure of the splenic tissues were affected by excessive fluoride. Nuclear dying, nuclear membrane dissolution, mitochondrial vacuolation, and endoplasmic reticulum dilation were observed. SCGE and MN assays showed that the nuclear DNA of splenocytes was damaged by fluoride treatment, and splenocyte apoptosis was exacerbated in the fluoride group. With damage to the splenocyte structure and DNA, the protein expression levels of IL-1ß, IL-2, IL-6, and TNF-α were significantly downregulated by exposure to fluoride. Excessive fluoride ingestion caused splenic pathological damage and abnormal cytokine expression in female rats.

Value of Ultrasonographic Features for Assessing Malignant Potential of Complex Cystic Breast Lesions.

OBJECTIVES: To assess the value of ultrasonography (US) features for determining the malignant potential of complex cystic lesions. METHODS: Seventy-nine complex cystic lesions were reviewed retrospectively. They were classified into four types according to US features in type I, the masses have a thick outer wall, thick internal septa, or both; in type II, the masses are an intracystic type with one or more discrete solid mural lesions within a cyst; in type III, the masses contain mixed cystic and solid components and are at least 50% cystic portion in a mass; in type IV, there are predominantly (at least 50%) solid masses with eccentric or central cystic foci. Positive predictive values were calculated for all types. RESULTS: The frequency of malignancy was higher among type III and IV lesions than among the other two types. Lesions with a diameter greater than or equal to 20 mm, margins not circumscribed, resistance index greater than or equal to 0.7, and axillary abnormal nodes had a high probability of malignancy. CONCLUSIONS: US is an important adjunct to evaluate the malignant potential of complex cystic lesions.

Effect of abdominal liposuction on sonographically guided high-intensity focused ultrasound ablation.

OBJECTIVES: The aim of this study was to evaluate the effect of abdominal liposuction on sonographically guided high-intensity focused ultrasound (HIFU) ablation. METHODS: A total of 10 women with uterine fibroids or adenomyosis who had received abdominal liposuction were analyzed after sonographically guided HIFU ablation. Of the 10 women, 6 had a diagnosis of uterine fibroids, and 4 had a diagnosis of uterine adenomyosis. All of them had a history of a horizontal-margin split-cesarean delivery. In addition, 26 women with a history of a single horizontal-margin split-cesarean delivery who had a diagnosis of uterine fibroids or adenomyosis but had not received liposuction were analyzed together as a control group. RESULTS: Of the 10 women, 1 woman with uterine fibroids developed local skin erythema after treatment; 1 women with uterine adenomyosis developed a skin burn after treatment; and the remaining women had obvious skin-burning pain during treatment. All women who had not received liposuction finished the treatment with no serious adverse events during or after treatment. The pain scores and incidence of skin-burning pain were significantly higher in the liposuction group than the control group (P= .021 and .038, respectively). CONCLUSIONS: Abdominal liposuction may increase the risk of skin burns during sonographically guided HIFU ablation.

[Ozone Pollution Characteristics and Sensitivity During the Ozone Pollution Days in Summer 2021 of Xinxiang City].

This study was based on the observation of volatile organic compounds (VOCs), conventional gaseous air pollutants, and meteorological parameters observed at the Xinxiang Municipal Party School site from June to August 2021. The ozone (O3) characteristics and sensitivity of O3 pollution days and the control strategy of its precursors were studied using an observation-based model (OBM). It was found that the meteorological conditions were characterized by high temperature, low humidity, and low pressure in O3-pollution days. The concentrations of O3 and its precursors all increased in the O3 pollution days. Oxygenated volatile organic compounds (OVOCs) and alkanes were the highest-concentration components of VOCs on O3 pollution days in Xinxiang, and OVOCs had the highest ozone formation potential (OFP) and hydroxyl (·OH) reactivity. According to the relative incremental reactivity (RIR) analysis, during the O3 pollution days in Xinxiang, O3sensitivity was in the VOCs-limited regime in June and in the transitional regime in July and August. Ozone production was more sensitive to alkenes and OVOCs. The RIR values of the precursors in June changed throughout the day, but O3 sensitivity remained the VOCs-limited regime. In July and August, O3 sensitivity was the VOCs-limited regime in the morning, transitional regime at noon, transitional and NOx-limited regime, respectively in the afternoon. By simulating different precursor-reduction scenarios, the results showed that the reduction of VOCs was always beneficial to the control of O3, whereas the reduction of NOx had little effect on the control of O3 and a risk of increasing O3.

Battacin (Octapeptin B5), a new cyclic lipopeptide antibiotic from Paenibacillus tianmuensis active against multidrug-resistant Gram-negative bacteria.

Hospital-acquired infections caused by drug-resistant bacteria are a significant challenge to patient safety. Numerous clinical isolates resistant to almost all commercially available antibiotics have emerged. Thus, novel antimicrobial agents, specifically those for multidrug-resistant Gram-negative bacteria, are urgently needed. In the current study, we report the isolation, structure elucidation, and preliminary biological characterization of a new cationic lipopeptide antibiotic, battacin or octapeptin B5, produced from a Paenibacillus tianmuensis soil isolate. Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of Escherichia coli and Pseudomonas aeruginosa are the pathogens most sensitive to battacin, with MICs of 2 to 4 µg/ml. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable to that of polymyxin B, the last-line therapy for infections caused by antibiotic-resistant Gram-negative bacteria. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane. Other studies indicate that battacin is less acutely toxic than polymyxin B and has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria.

Identification and functional analysis of gene cluster involvement in biosynthesis of the cyclic lipopeptide antibiotic pelgipeptin produced by Paenibacillus elgii.

BACKGROUND: Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a ß-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. RESULTS: A potential pelgipeptin synthetase gene cluster (plp) was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs), with one, seven, and one module(s), respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1) provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. CONCLUSIONS: In this study, a gene cluster (plp) responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.

Fluoride exposure cause colon microbiota dysbiosis by destroyed microenvironment and disturbed antimicrobial peptides expression in colon.

Colon microenvironment and microbiota dysbiosis are closely related to various human metabolic diseases. In this study, a total of 72 healthy female mice were exposed to fluoride (F) (0, 25, 50 and 100 mg/L F-) in drinking water for 70 days. The effect of F on intestinal barrier and the diversity and composition in colon microbiota have been evaluated. Meanwhile, the relationship among F-induced colon microbiota alterations and antimicrobial peptides (AMPs) expression and short-chain fatty acids (SCFAs) level also been assessed. The results suggested that F decreased the goblet cells number and glycoprotein expression in colon. And further high-throughput 16S rRNA gene sequencing result demonstrated that F exposure induced the diversity and community composition of colonic microbiota significantly changes. Linear Discriminant Analysis Effect Size (LEfSe) analysis identified 11 predominantly characteristic taxa which may be the biomarker in response to F exposure. F-induced intestinal microbiota perturbations lead to the significantly decreased SCFAs levels in colon. Immunofluorescence results showed that F increased the protein expression of interleukin-17A (IL-17A) and IL-22 (P < 0.01) and disturbed the expression of interleukin-17 receptor A (IL-17RA) and IL-22R (P < 0.05 or P < 0.01). In addition, the increased expression of IL-17A and IL-22 cooperatively enhanced the mRNA expression of AMPs which response to F-induced microbiota perturbations. Collectively, destroyed microenvironment and disturbed AMPs are the primary reason of microbiota dysbiosis in colon after F exposure. Colonic homoeostasis imbalance would be helpful for finding the source of F-induced chronic systemic diseases.

Hepatocellular carcinoma with type II-III portal vein tumour thrombosis: treatment using transarterial chemoembolisation and microwave ablation.

OBJECTIVE: To evaluate the use of transarterial chemoembolisation (TACE) combined with microwave ablation (MWA) to treat patients with hepatocellular carcinoma (HCC) and type Ⅱ-Ⅲ portal vein tumour thrombosis (PVTT) intolerant to targeted drug (TG) therapy. METHODS: A total of 18 patients with HCC and type Ⅱ-Ⅲ PVTT intolerant to TG were enrolled between June 2015 and December 2019, who were treated with TACE + MWA (MWA group). 24 patients were treated with TACE + TG (TG group; control cohort). Time to progression and overall survival (OS) were analysed along with the incidence of adverse events. RESULTS: The median follow-up time was 19.0 months (9.0-32.0 months). The median OS was 17.0 months (8.3-29.3 months; MWA group) and 13.5 months (5.5-22.5 months; TG group) and was not significantly different. The 1- and 2 year OS was also comparable (MWA group: 66.7%, 44.4% vs Target group: 41.7%, 29.2%). Time to progression showed no distinct differences (MWA group: 11.5 months; TG group: 9.0 months) between the two groups. Moreover, the incidence of major Grade 3-4 adverse events in the MWA group (5.6%) was similar to those in the TG group (8.3%). CONCLUSION: TACE + MWA and TACE + TG were comparable in their safety and efficacy in patients with HCC, type Ⅱ-Ⅲ PVTT, and intolerance to TG. ADVANCES IN KNOWLEDGE: TACE + MWA can be used as a palliative treatment alternative for TACE + TG in patients with HCC, type Ⅱ-Ⅲ PVTT, and intolerance to TG.

Mitochondrial respiratory chain damage and mitochondrial fusion disorder are involved in liver dysfunction of fluoride-induced mice.

Our previous study showed that excessive fluoride (F) intake can induce liver dysfunction. The aim of this study was to investigate the mechanisms of F-induced mitochondrial damage resulting in liver dysfunction. Damaged mitochondrial ultrastructure and state of liver cells were estimated by TEM, TUNEL staining and BrdU measurement. The ROS level and ATP content in the liver tissue were measured by ELISA kit. Meanwhile, optic atrophy (OPA1), mitofusin-1 (Mfn1), NDUFV2, SDHA, CYC1, and COX Ⅳ expression levels were measured through real-time PCR and Western-blot. Results showed that the ROS level increased, thereby resulting in mitochondrial ultrastructure damage and abundant liver cells presented evident apoptotic characteristics after F treatment. Decreased ATP content and the abnormal expression of OPA1, Mfn1, NDUFV2, SDHA, CYC1, and COX Ⅳ of the liver tissue were observed. In conclusion, excessive F-induced mitochondrial respiratory chain damaged and mitochondrial fusion disorder resulted in liver dysfunction.

Mitochondrial respiratory chain complex abnormal expressions and fusion disorder are involved in fluoride-induced mitochondrial dysfunction in ovarian granulosa cells.

Excessive fluoride intake has a strong female reproductive toxicity, which can result in follicular developmental dysplasia and decrease oocytes developmental potential. The underlying mechanisms of fluoride-induced mitochondrial dysfunction in ovarian granulosa cells remain largely unknown. In this study, the ultrastructure changes of mitochondria and DNA damage in ovarian granulosa cells were observed under transmission electron microscope and TUNEL staining. Then, the ATP content and ROS level in granulosa cells were measured. The expression of mitochondrial fusion proteins and mitochondrial respiratory chain complexes, including OPA1 and Mfn1, and NDUFV2, SDHA and CYC1, in the ovarian tissues were measured by immunohistochemistry, Western blot and Quantitative real-time PCR analyses. The expression of ATP5j and ATP5h in the ovarian tissues was also measured. Results show that fluoride treatment considerably damages mitochondrial ultrastructure and enhances the apoptosis of granulosa cells. The ATP content greatly decreased, whereas the ROS level increased after fluoride treatment. The expression level of Mfn1 in the ovarian tissue was up-regulated, whereas OPA1 expression had no significant change. The expression levels of NDUFV2, SDHA and CYC1 were considerably up-regulated, and the expression of ATP5j and ATP5h were down-regulated after fluoride treatment. In summary, the damage in the mitochondrial ultrastructure, ATP content decrease, ROS level increase and the abnormal expression of OPA1, Mfn1, NDUFV2, SDHA, CYC1, ATP5j and ATP5h in ovary tissue are closely associated with fluoride-induced mitochondrial dysfunction, which might be responsible for the follicular developmental dysplasia and the potential decrease in oocyte development induced by fluoride in female mice.

Effect of Fluoride on Small Intestine Morphology and Serum Cytokine Contents in Rats.

This study aimed to determine the effect of excessive fluoride (F) on the morphological characteristics of the small intestine and the contents of serum cytokines in rats. A total of 48 3-week-old healthy female Sprague-Dawley rats were randomly divided into four groups (n = 12). The control group was given deionized distilled water, while the F treatment groups were treated with water containing 25, 50, and 100 mg F-/L. After 70 days of treatment, the duodenum, the jejunum, and the ileum were collected to measure the developmental parameters and the distribution of intestinal glycoproteins, goblet cells, and mast cells through Pannoramic Viewer, Periodic Acid-Schiff (PAS) staining, Alcian blue and periodic acid-Schiff (AB-PAS) staining, and toluidine blue staining, respectively. The contents of cytokines, namely, interleukin (IL)-1ß, IL-2, IL-6, and tumor necrosis factor (TNF)-α, in serum were detected via enzyme-linked immunosorbent assay (ELISA). Results showed that the villus height, crypt depth, villus height to crypt depth ratio, goblet cells, glycoproteins, and mast cells of the small intestine significantly decreased (P < 0.05 or P < 0.01) in the F treatment group. The contents of IL-1ß, IL-2, IL-6, and TNF-α were significantly lower in the F treatment group than in the control group (P < 0.05 or P < 0.01). In summary, excessive F intake impaired intestinal development and immune function by decreasing the developmental parameters and the distribution of immune cells, glycoproteins, and cytokines.

PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells.

To investigate the mechanisms of fluoride-induced apoptosis, a fluoride-induced C2C12 skeletal muscle cell (C2C12 cell) model was established in this study, and the viability of the C2C12 cells was measured using an MTT assay. Cell morphological changes were observed via haematoxylin and eosin staining and transmission electron microscopy. Apoptosis was monitored through Hoechst staining. The mRNA and protein expression of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and caspase-9 were detected through real-time PCR and western blotting, respectively. The results showed that the survival rates of C2C12 cells decreased gradually with an increasing fluoride doses. The C2C12 cell structure was seriously damaged by fluoride, presenting with pyknosis, mitochondrial ridge disruption and swollen endoplasmic reticulum. Furthermore, the expression of mRNA in PI3K, BAD, Bcl-2, Bax and caspase-9 were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 was markedly decreased (P < 0.01). The expression of protein in BAD, Bcl-2 and Bax were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 and P-AKT1 was markedly decreased (P < 0.01). In conclusion, fluoride-induced apoptosis in C2C12 cells is related to the PI3K/AKT signaling pathway.

Mitochondria-Mediated Pathway Regulates C2C12 Cell Apoptosis Induced by Fluoride.

This study was designed to investigate the mechanisms of excessive fluoride-induced apoptosis via mitochondria-mediated pathway in skeletal muscle cells (C2C12 cells). C2C12 cells were cultured with the fluoride concentrations (0, 1, and 2.5 mmol/L) for 48 h. The morphology and ultrastructural changes of C2C12 cells were observed using a light microscope and transmission electron microscope (TEM). The protein expression levels of apoptosis factors, including Bax, Bcl-2, cytochrome c (Cyt c), caspase-3, and caspase-9, were measured using real-time polymerase chain reaction (real-time PCR) and immunocytofluorescence. The morphology and ultrastructure of C2C12 cells were seriously damaged by fluoride at 1 and 2.5 mmol/L doses, including swollen mitochondria, vacuolization, ridge breakage, and disappearance of the nuclear membrane. Simultaneously, compared with the control group, the expression levels of Bax, Bcl-2, Cyt c, caspase-3, and caspase-9 were up-regulated after fluoride treatment. Excessive fluoride damages the ultrastructure in mitochondria, leading to the release of Cyt c from the mitochondria to cytoplasm in C2C12 cells; thereby, activated caspases cascade apoptosis process through a mitochondria-mediated pathway.

JNK/STAT signalling pathway is involved in fluoride-induced follicular developmental dysplasia in female mice.

Excessive fluoride (F) intake decreases the development of potential oocytes by inducing oxidative stress and apoptosis in female mice in our previous study. This study aims to investigate the underlying mechanisms of F-induced follicular developmental dysplasia. Pathomorphological changes in the ovary tissues were observed under light and transmission electron microscopes. DNA damage and proliferation in granulosa cells were analysed by TUNEL staining and BrdU measurement. The protein expression of cell proliferation related regulatory factors including JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the ovary tissues was measured by immunohistochemistry and Western blot analyses. Results indicated that the structure of granulosa cells in the ovary was seriously damaged by excessive F, evident by the swollen endoplasmic reticulum, mitochondria with vacuoles and nucleus shrinkage. F treatment also considerably enhanced the apoptosis and inhibited the proliferation of granulosa cells. The number of granulosa cells around the oocyte decreased after F treatment. The expression levels of STAT3, CDK2, CDK4 and Ki67 in the ovary tissues were up-regulated, and STAT5 and PCNA did not change significantly after F treatment, whereas JNK expression was down-regulated with increasing F dose. In summary, changes in the expression levels of JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the JNK/STAT signalling pathway are involved in F-induced follicular dysplasia in the ovary.

Ca2+ metabolic disorder and abnormal expression of cardiac troponin involved in fluoride-induced cardiomyocyte damage.

Our previous study indicated that excessive fluoride (F) induces ATP5J and ATP5H proactive expression by interfering cardiomyocyte mitochondrial dysfunction in mice. This study aimed to investigate underlying mechanisms of F¯ induced damage to cardiomyocytes. A total of 100 mg/L F¯ was added to distilled water to treat Kunming mice for 70 days. Pathological and morphological changes in myocardial tissues were observed under transmission electron microscope and light microscope. Content of ATP and ATP enzyme distributed in cardiomyocytes were determined by fluorescence and ATP enzyme staining. Expression levels of troponin (Tn) C, TnI, TnT and tropomyosin (TPM) were measured by immunofluorescence, western blot, and real-time polymerase chain reaction. Contents of Ca2+ in blood, myocardial cells and faeces were also detected by confocal microscopy and ethylenediaminetetraacetic acid. Using 100 mg/L F¯ resulted in nuclaer enrichment, the myocardial fibre breakage and mitochondrial lysis. Following mitochondrial structure damage, contents of ATP and ATP enzyme significantly decreased in the fluoride group. Expression levels of TnT and TnI were significantly down-regulated, whereas that of TPM was up-regulated. Content of Ca2+ in cardiomyocytes of fluoride group visibly increased. Interestingly, contents of Ca2+ in blood and faeces decreased. These findings reveal that excessive F ingestion induces Ca2+ metabolic disorder, and an abnormal expression of cardiac Tn are involved in F-induced cardiomyocyte damage.

Positive PCNA and Ki-67 Expression in the Testis Correlates with Spermatogenesis Dysfunction in Fluoride-Treated Rats.

The present study aimed to evaluate the effect of fluoride (F) on spermatogenesis in male rats. F- at 50 and 100 mg/L was administered for 70 days, after which the testicular and epididymis tissues were collected to observe the histopathological structure under a light microscope. The ultrastructure of the testis and sperm was also examined via transmission electron microscopy. The apoptosis of spermatogenic cells was measured through terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of proliferation factors, namely, proliferating cell nuclear antigen (PCNA) and Ki-67, in the testicular and epididymis tissues, were assayed through immunohistochemistry. F- at 50 and 100 mg/L significantly damaged the structure of the testis and epididymis, and the testis and sperm ultrastructure exhibited various changes, including mitochondrial swelling and vacuolization, and apsilated and raised sperm membrane. F treatment significantly increased spermatogenic cell apoptosis in the testis. PCNA (P < 0.01) and Ki-67 (P < 0.01) also presented positive expression in the testis. By comparison, no significant changes occurred in the epididymis. In summary, excessive F intake results in spermatogenesis dysfunction by damaging the testicular structure and inducing spermatogenic cell apoptosis in male rats. The positive expression level of PCNA and Ki-67 was a good response to spermatogenesis dysfunction.

Combination of 4-hydroperoxy cyclophosphamide and methotrexate inhibits IL-6/sIL-6R-induced RANKL expression in fibroblast-like synoviocytes via suppression of the JAK2/STAT3 and p38MAPK signaling pathway.

Although conventional combination therapy is effective for most patients with rheumatoid arthritis (RA), many still do not respond to current therapies. Therefore, novel combination regimens that better target cellular processes involved in RA pathogenesis are required. Preliminary studies have demonstrated the beneficial effects of a combination of cyclophosphamide (CTX) and methotrexate (MTX) in models of RA. Using western blotting, real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and immunofluorescent staining, we demonstrated that the combination of 4-hydroperoxy CTX (4-H-CTX) and MTX inhibited the expression of receptor activator of nuclear factor-κB ligand (RANKL) in fibroblast-like synoviocytes (FLS) treated with the interleukin (IL)-6/soluble IL-6 receptor (sIL-6R) complex. To elucidate the mechanisms underlying this effect, we treated RA-FLS with the JAK2/STAT3 inhibitor AG490 or p38MAPK inhibitor SB203580. The results showed that IL-6/sIL-6R-induced RANKL upregulation required phosphorylation-mediated activation of STAT3 and p38 signaling, and that 4-H-CTX and/or MTX inhibited RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing JAK2/STAT3 and p38MAPK signaling. This study demonstrated for the first time the inhibitory effects of 4-H-CTX and MTX on RANKL expression in IL-6/sIL-6R-stimulated FLS via suppression of STAT3 and p38MAPK phosphorylation. These results identify promising therapeutic agents that might have clinical applications in patients with RA who are at high risk of bone erosion or do not respond well to conventional therapy.

Fluoride-induced oxidative stress and apoptosis are involved in the reducing of oocytes development potential in mice.

The present study was conducted to investigate the mechanisms of excessive-fluoride-induced reduction of oocyte development potential in mice. The development morphology of oocyte and the changes of pathomorphology in ovary were observed. The protein expression levels of apoptosis factors, including Bax, Bcl-2, casepase-3, casepase-9 and cytochrome c, and the mRNA expression levels of antioxidant enzymes, including SOD1, GSH-Px1, CAT and inducible nitric oxide synthase were measured by Western blot and real-time PCR, respectively. DNA damage in the ovary was analysed by single cell gel electrophoresis and TUNEL staining. Results indicated that the structure and function of ovarian cells were seriously damaged, followed, the development potential of oocyte was reduced by excessive fluoride. The expression levels of apoptosis factors were up-regulated and antioxidant enzymes were significantly down-regulated. Meanwhile, the contents of ROS, MDA, NO and iNOS were significantly increased. Whereas, the activities of SOD1, GSH-Px1 and CAT was significantly decreased compared with the control group. Simultaneously, the results of DNA analysis indicated that the tail length and tailing ratio of ovarian cells were significantly increased in the fluoride group. In summary, the results provided compelling evidence that excessive fluoride intake can reduce the development potential of oocyte by inducing oxidative stress and apoptosis in the ovary of female mice.