Inhibited Alkaline Phosphatase Activity Research Articles

Effects of Empagliflozin on Vascular and Skeletal Mineralization in Hyperlipidemic Mice

Cardiovascular disease and osteoporosis, major causes of morbidity and mortality, are associated with hyperlipidemia. Recent studies show that empagliflozin (EMPA), an inhibitor of sodium-glucose cotransporter-2 (SGLT2), improves cardiovascular health. In preclinical animal studies, EMPA mitigates vascular calcification in the males but its effects in the females are not known. Thus, we used female mice to test the effects of EMPA on calcification in the artery wall, cardiac function, and skeletal bone. By serial in vivo microCT imaging, we followed the progression of aortic calcification and bone mineral density in young and older female Apoe−/− mice fed a high-fat diet with or without EMPA. The two different age groups were used to compare early vs. advanced stages of aortic calcification. Results show that EMPA treatment increased urine glucose levels. Aortic calcium content increased in both the controls and the EMPA-treated mice, and EMPA did not affect progression of aortic calcium content in both young and older mice. However, 3-D segmentation analysis of aortic calcium deposits on microCT images revealed that EMPA-treated mice had significantly less surface area and volume of calcified deposits as well as fewer numbers of deposits than the control mice. To test for direct effects on vascular cell calcification, we treated murine aortic smooth muscle cells with EMPA, and results showed a slight inhibition of alkaline phosphatase activity and inflammatory matrix calcification. As for skeletal bone, EMPA-treated mice had significantly lower BMD than the controls in both the lumbar vertebrae and femoral bones in both young and older mice. The findings suggest that, in hyperlipidemic female mice, unlike males, SGLT2 inhibition with empagliflozin does not mitigate progression of aortic calcification and may even lower skeletal bone density.

Interferon Regulatory Factor 4 (IRF4) Plays a Key Role in Osteoblast Differentiation of Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (PMOP) is a prevalent disease, which features decreased bone mass, bone weakness and deteriorated bone microstructure in postmenopausal women. Although many factors have been revealed to contribute to the occurrence of PMOP, its mechanism remains undefined. This work aimed to identify significant changes in gene expression during PMOP formation and to examine the most valuable differential genes in postmenopausal osteoporosis versus the control group. The GSE68303 dataset that contains 12 ovariectomize (OVX) experimental and 11 sham groups was downloaded and analyzed. The results indicated that interferon regulatory factor 4 (IRF4) might be a hub gene in the development of postmenopausal osteoporosis. Western blot and immunohistochemistry were carried out to evaluate IRF4 levels in thoracic vertebra extracts from OVX and Sham mice. To assess IRF4's impact on osteogenic differentiation in postmenopausal bone marrow mesenchymal stem cells (BM-MSCs), IRF4 overexpression (OV-IRF4) and knockdown (Sh-IRF4) plasmids were constructed. The results showed that comparing with the sham group, bone samples from the OVX group showed higher IRF4 expression. Alkaline phosphatase (ALP) staining revealed that IRF4 overexpression significantly inhibited ALP activity, while IRF4 knockdown promoted ALP activity in BM-MSCs. Simvastatin-treated OVX mice showed increased total bone volume/total tissue volume (BV/TV) and elevated Runx2 expression by immunohistochemical staining compared with the OVX group. This study demonstrated that IRF4 is associated with OVX induced osteoporosis, it can regulate bone stability by inhibiting the osteogenic differentiation BM-MSCs. This study may help enhance our understanding of the molecular mechanism of PMOP formation, providing new insights into estrogen defiance induced osteoporosis.

CircHIPK3 relieves vascular calcification via mediating SIRT1/PGC-1α/MFN2 pathway by interacting with FUS

BackgroundCircular RNAs (circRNAs) have been reported to regulate the biological processes of human diseases. CircHIPK3 has been implicated in vascular calcification, but the downstream regulatory mechanisms remain unclear. Our study aimed to understand the regulatory function of circHIPK3 in vascular calcification.MethodsCircHIPK3 expression in atherosclerosis (AS) serum samples and vascular smooth muscle cells (VSMCs) calcification model was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The binding relationships between fused in sarcoma (FUS) and circHIPK3 or sirtuin 1 (SIRT1) were verified by RNA immunoprecipitation (RIP) assay and RNA pull-down assays. Alkaline phosphatase (ALP) activity and alizarin red staining assays were performed to evaluate the biological effect of β-glycerophosphate (β-GP) and circHIPK3 on calcium deposition. qRT-PCR and western blot assays were used to examine the effect of β-GP, circHIPK3, SIRT1, mitofusin 2 (MFN2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) on VSMCs calcification and the expression of calcification-related proteins.ResultsIn AS serum samples and VSMCs calcification model, the expression of circHIPK3 was significantly reduced. CircHIPK3 overexpression inhibited ALP activity and calcium deposition in β-GP-induced VSMCs. Moreover, circHIPK3 could recruit FUS to further stabilize SIRT1 mRNA. CircHIPK3 promoted MFN2 expression to alleviate VSMCs calcification via activating SIRT1/PGC-1α signaling.ConclusionThe positive regulation of circHIPK3/FUS/SIRT1/PGC-1α/MFN2 signaling pathway contributed to the alleviate VSMCs calcification, revealing a novel regulatory axis for vascular calcification.

Assessing the molluscicidal and biochemical activities of New-Fort®, an inorganic fertilizer against Theba pisana snails

Terrestrial snails are a significant issue in agricultural production worldwide. The use of nitrogen - phosphorus - potassium (NPK) based fertilizers played an important role in meeting the food demand throughout the world, so its effectiveness against land snails needs to be investigated. This study was conducted to evaluate toxic lethal effect of New-Fort®, an inorganic NPK based fertilizer, in the field for 3, 7 and 10 days and in the laboratory for 24, 48 and 72 h against Theba pisana snails. Also, the impact of its sub-lethal doses (1/10, 1/5, 1/4 and 1/2 of 48 h-LD50) on biochemical parameters were determined under laboratory conditions. The results showed that the snails percent reduction in the field were 21.4, 61.0 and 80.0 % after 10 days' application of quarter, half and one field rate and the values of LD50 in the laboratory were 4.94, 4.56 and 4.24 mg/g b.w at 24, 48 and 72 h, respectively. New-Fort® sub-lethal doses caused a significant inhibition in catalase, γ-glutamyl transferase and acetylcholinesterase activities. It also elicited a significant elevation in glutathione S-transferase activity post exposure to 1/10 and 1/5 of LD50, whereas an opposite effect was occurred after exposure to 1/4 and 1/2 of LD50. Lipid peroxidation level was reduced in snails treated with 1/10 and 1/5 of LD50, whereas it increased in 1/4 and 1/2 of LD50- treated snails. Moreover, a significant inhibition in alkaline phosphatase activity at all tested doses, with the exception of 1/2 of LD50 was observed. An increase in alanine aminotransferase and aspartate aminotransferase activities were occurred after all tested doses exposure. Our findings highlighted on how biochemical changes can be exploited to better understand the mechanisms underlying New-Fort® fertilizer toxicity against the land snail, T. pisana, as well as how to benefit from NPK fertilizers application in snail control.

Development of a novel apigenin prodrug programmed for alkaline-phosphatase instructed self-inhibition to combat cancer

Elevated levels of alkaline phosphatase (ALP) in the tumor microenvironment (TME) are a hallmark of cancer progression and thus inhibition of ALP could serve as an effective approach against cancer. Herein, we developed a novel prodrug approach to tackle cancer that bears self-inhibiting alkaline phosphatase-responsiveness properties that can enhance at the same time the solubility of the parent compound. To probe this novel concept, we selected apigenin as the cytotoxic agent since we first unveiled, that it directly interacts and inhibits ALP activity. Consequently, we rationally designed and synthesized, using a self-immolative linker, an ALP responsive apigenin-based phosphate prodrug, phospho-apigenin. Phospho-apigenin markedly increased the stability of the parent compound apigenin. Furthermore, the prodrug exhibited enhanced antiproliferative effect in malignant cells with elevated ALP levels, compared to apigenin. This recorded potency of the developed prodrug was further confirmed in vivo where phospho-apigenin significantly suppressed by 52.8% the growth of PC-3 xenograft tumors. Communicated by Ramaswamy H. Sarma

A Turn-On Fluorescent Assay for Glyphosate Determination Based on Polydopamine-Polyethyleneimine Copolymer via the Inner Filter Effect

The threat of glyphosate to food safety has attracted widespread attention. Consequently, it is highly urgent to develop a sensitive and accurate method for glyphosate detection. Herein, a turn-on fluorescent method for glyphosate detection using polydopamine-polyethyleneimine (PDA-PEI) copolymer as a fluorescent probe and p-nitrophenylphosphate (PNPP)/alkaline phosphatase (ALP) as a fluorescence quenching system is developed. The PDA-PEI copolymer was prepared by a one-pot method under mild condition, and its fluorescence kept almost unchanged after storing in a refrigerator for one month. ALP catalyzed the hydrolysis of PNPP to p-nitrophenol (PNP) that caused the fluorescence quenching of PDA-PEI copolymer via the inner filter effect. However, glyphosate inhibited ALP activity, thereby preventing the formation of PNP and restoring the fluorescence signal. Under the optimized conditions, the fluorescence of PDA-PEI copolymer depended on glyphosate concentrations ranging from 0.2 to 10 μg/mL with a detection limit of 0.06 μg/mL. Moreover, this assay was applied to detect glyphosate in real samples using the standard addition method. The recoveries were in the range from 88.8% to 107.0% with RSD less than 7.78%. This study provides a novel insight for glyphosate detection and expands the applications of fluorescent copolymer.

SOX9 Inhibits the Progression of Osteonecrosis of the Femoral Head via the Activation of the Wnt/Beta-Catenin Pathway

Background: In this study, we aimed to explore whether the SRY-box transcription factor 9 (SOX9) can play protective roles against the occurrence and development of osteonecrosis of the femoral head (ONFH) by regulating the proliferation, apoptosis, and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) via the Wnt/β-catenin pathway. Methods: We used 1600 mg of glucocorticoid (GC) to induce hBMSCs to establish an ONFH cell model and performed various experiments. Reverse transcription-quantitative polymerase chain reaction and western blotting assays were used to determine the expression levels of SOX9 and osteoblast markers, such as the RUNX family transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osterix, Wnt3a, and β-catenin. An ALP detection kit was used to measure the ALP activity. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to determine the cell viability. Results: GC treatment decreased the expression levels of RUNX2, ALP, and osterix, suppressed ALP activity, and inhibited SOX9 expression. SOX9 overexpression promoted GC-induced cell proliferation and decreased cell apoptosis. Additionally, hBMSCs were transfected with SOX9-small interfering RNA in GC treatment, and SOX9 knockdown was found to suppress the osteogenic differentiation of cells and decrease their viability. Conclusion: Our results revealed that SOX9 is related to the Wnt/β-catenin pathway in ONFH. Moreover, SOX9 participated in ONFH development by activating the Wnt/β-catenin pathway.

Toxicity of single and ternary mixtures of heavy metals and 2,4-dichlorophenoxyacetic acid to Chlorella vulgaris alkaline phosphatase activity

The influence of mixtures of metals and pesticide on the activity of algal hydrolytic enzyme alkaline phosphatase was investigated. Toxicity of unary and ternary combinations of Copper (Cu2+), Zinc (Zn2+), Lead (Pb2+), Chromium (Cr2+), Cadmium (Cd2+) ions and 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed via inhibition of phosphatase enzyme activity of Chlorella vulgaris. The effects of the amalgamated ternary mixtures were studied in their fixed percentage ratio of 20:40:40 and 40:30:30. Results obtained showed that all the unary heavy metal ions were toxic to algal phosphatase activity, with a concentration dependent inhibition of alkaline phosphatase activity. The analysis of eco-toxicity concentration (EC50) of toxicant ternary mixtures of metals/ pesticides mixtures in 20:40:40 ratios show that the ternary mixtures 2,4-D/Pb2+/Cd2+and 2,4-D/Zn2+/Cd2+ were more toxic, followed by 2,4-D/Cu2+/Cr2+, 2,4-D/Pb2+/Zn2+, 2,4-D/Cu2+/Zn2+ and 2,4-D/Cu2+/Cd2+. Whereas among the 40:30:30 mixture ratios 2,4-D/Pb2+/Cd2+ and 2,4-D/Zn2+/Cd2+ were most toxic. The decline in the trend of their toxicity strength was followed by 2,4-D/Cu2+/Cr2+, 2,4-D/Cu2+/Zn2+, 2,4-D/Cu2+/Cd2+, 2,4-D/Cu2+/Pb2+ and 2,4-D/Pb2+/Zn2+. In addition, the least toxic amongst them were the 2,4-D/Pb2+/Cr2+, 2,4-D/Zn2+/Cr2+ and 2,4-D/Cr2+/Cd2+ ternary mixtures. Toxicity index analysis of the mixture interaction effect showed that the mixtures of heavy metals with 2,4, D were mostly antagonistic with toxic index (TI) >>1, However, exceptions were the 2,4-D/Pb2+/Cd2+ and 2,4-D/Pb2+/Cd2+mixture which were additive and synergistic with TI=1 and TI<1 respectively. From the foregoing study, the toxicity of the mixtures exhibited largely sigmoidal relationship, increased composition of heavy metal in the mixture resulted in a potentiation of 2,4-D toxicity. These results indicate that mixtures of heavy metals with largely non-toxic 2,4-D may pose ecological risk to freshwater microalgae Chlorella vulgaris, presenting antagonistic, additive and synergistic toxicity interactions based on their relative occurrence in the mixture.

The Examination of Remodeling Processes of Hard Periodontal Tissues in Rats with Disorders of Pancreatic Incretory Function

Topicality. Pancreatic hormones play an important role in the process of remodeling of hard periodontal tissues. Violations of carbohydrate metabolism that occur under conditions of isolated insulin resistance (IR) and in combination with iodine deficiency (ID) can be the cause of dysmetabolic disorders of mineralization/demineralization physiological system of tooth-maxillary complex.
 The aim. To study the changes of remodeling processes of hard periodontal tissues in rats with isolated IR and impaired glucose tolerance against the background of ID.
 Materials and methods. The examination was carried on 90 male rats, which were divided into three groups: control (intact animals), group of rats with IR under conditions of adequate iodine supply, and with IR against the background of ID. The system of carbohydrate metabolism was studied by the level of insulin in blood serum, glucose and glycosylated hemoglobin (HbA1c) of blood with the following calculation of HOMA-IR index. The processes of mineralization/demineralization were detected by the content of calcium, magnesium, zinc, manganese, copper in cementum of tooth root and alveolar process, and by the activity of acid and alkaline phosphatases in blood serum.
 Results. Keeping of animals on a high-fructose diet led to the development of carbohydrate metabolism disorders (increase blood glucose and HbA1c levels, blood serum insulin, HOMA-IR index) and changes of remodeling processes in hard periodontal tissues (decrease the level of calcium, magnesium and manganese against the background of increase the zinc content in cementum of tooth root; decrease the content of macroelements in alveolar process; activation of acid phosphatase against the background of inhibition of alkaline phosphatase activity). The development of combined endocrinopathy was accompanied by more pronounced changes of studied parameters.
 Conclusions. The violation of glucose tolerance against the background of ID slows down the mineralization processes of hard periodontal tissues mainly due to the intensification of osteoresorptive processes.
 Keywords: acid and alkaline phosphatases; calcium homeostasis; bioelement panel of tooth-maxillary system; insulin resistance; iodine deficiency.

Unexpected Stability of a Prodrug to Enzymatic Hydrolysis within a Hydrated HPMC Matrix Tablet.

The uptake of alkaline phosphate present in dissolution medium into a hydrating hydroxypropyl methylcellulose matrix tablet and that its activity was retained therein was demonstrated. This presents a risk to the stability of prodrugs that are substrates of this enzyme such as phosphonooxymethyl derivative prodrugs. It was found that fostemsavir, a phosphonooxymethyl derivative prodrug being developed for the treatment of HIV-1 infection, was unexpectedly resistant to hydrolysis within a hydrated HPMC matrix when subjected to drug release testing in media containing alkaline phosphatase. Studies indicated that this was not due to microenvironmental pH effects, osmolality effects or effective phosphate concentration effects associated with the presence of the prodrug. That the prodrug and not its parent could affect enzyme activity in a concentration dependent manner, and that another phosphate ester prodrug fosphenytoin did not inhibit alkaline phosphatase activity within a hydrated HPMC matrix suggested that the unexpected stability of the HIV-1 therapy prodrug may be associated with the ability of the phosphate group-containing compound itself to inhibit the enzyme at the concentrations it exists at in the hydrated dosage form and so enables the development of the compound in this type of dosage form.

LncRNA MALAT1 promotes osteogenic differentiation through the miR-217/AKT3 axis: A possible strategy to alleviate osteoporosis.

Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) are associated with the development of osteoporosis. The present study aimed to investigate the effect of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on osteogenic differentiation in osteoporosis. MALAT1 levels were detected by a real-time polymerase chain reaction (RT-qPCR). Moreover, the levels of osteogenic differentiation-related factors (Bmp4, Col1a1 and Spp1) were measured by a RT-qPCR and western blotting. Alkaline phosphatase (ALP) activity was detected using an ALP staining assay. MALAT1 levels were downregulated in hindlimb unloading mice and simulated in microgravity (MG) treated MC3T3-E1 cells. Moreover, MG treatment induced the downregulation of the expression of ALP, BMP4, Col1a1 and Spp1, whereas overexpression of MALAT1 abolished the downregulation. MG also inhibited ALP activity, whereas MALAT1 reversed the effect. Furthermore, miR-217 was identified as a target of MALAT1, and AKT3 was verified as a target of miR-217. Overexpression of miR-217 rescued the promotion of osteogenic differentiation induced by MALAT1 in MG treated cells. Knockdown of AKT3 abolished the facilitation of osteogenic differentiation induced by downregulation of miR-217. MALAT1 promotes osteogenic differentiation through regulating miR-217/AKT3 axis, suggesting that MALAT1 is a potential target with respect to alleviating osteoporosis.

Paper-based multicolor sensor for on-site quantitative detection of 2,4-dichlorophenoxyacetic acid based on alkaline phosphatase-mediated gold nanobipyramids growth and colorimeter-assisted method for quantifying color

On-site quantitative analysis of pesticides is important for food safety. Colorimetric gold nanobipyramids (AuNBPs) sensors are powerful methods for on-site detection. However, a single quantitative method and the instability of AuNBPs in solution limit the practicability of those sensors. Here, a paper-based multicolor AuNBPs sensor involved a colorimeter-assisted method for quantifying color was developed for quantitative detection of 2,4-dichlorophenoxyacetic acid (2,4-D), a common herbicide. The novelty of this study lies in developing a general paper-based quantitative on-site method (PQOM) for colorimetric AuNBPs sensors. Firstly, a paper-based analytical device (PAD) consisting of a nylon membrane, absorbent cotton layers, and two acrylic plates was fabricated to deposit AuNBPs. We demonstrated the PAD could improve the stability of AuNBPs and the detection sensitivity of AuNBPs sensors. Then, a handheld colorimeter was first used to quantify the color change of AuNBPs on the PAD based on the CIELab color space. Finally, as proof of concept, the PQOM was successfully employed to quantify 2,4-D by combining with an alkaline phosphatase-mediated AuNBPs growth method. In this method, 2,4-D specifically inhibited alkaline phosphatase activity to suppress the generation of l-ascorbic acid, thereby mediating AuNBPs growth. The developed sensor exhibited seven 2,4-D concentration-related colors and detected as low as 50 ng mL−1 2,4-D by naked-eye observation and 18 ng mL−1 2,4-D by a colorimeter. It was applied to detect 2,4-D in the spiked rice and apple samples with good recovery rates (91.8–112.0%) and a relative standard deviation (n = 5) < 5%. The success of this study provides a sensing platform for quantifying 2,4-D on site.

The calcium supplementation effect of calcium-binding oligopeptides from bonito (Auxis thazard) hydrolysate in rats

Sixty Ca-deficient male Wistar rats were divided into six groups to investigate the effect of a calcium-binding bonito hydrolysate (PBH-Ca) on calcium bioavailability in rats, including the normal group, the control groups (PBH + CaCO3 group and the CaCO3 group), and three PBH-Ca groups with doses of 2,000 (PBH-Ca-H group), 1,000 (PBH-Ca-M group), and 500 (PBH-Ca-L group) mg/kg body wt/day. Each group was given the corresponding drug by intragastric administration for 4 weeks. The indices measured included the rate of increase in body weight and body length, apparent calcium absorption rate, serum calcium, serum phosphorus, alkaline phosphatase (ALP) activity, bone volume fraction (BV/TV), bone mineral density (BMD), and trabecular microstructure of the rats scanned by micro-computed tomography. The results showed that PBH-Ca promoted the growth of rats, significantly increased the apparent calcium absorption rate and the number of bone trabeculae, stabilized serum calcium and serum phosphorus content in serum, significantly inhibited ALP activity, decreased the separation of bone trabeculae, accelerated bone growth of rats, and raised the BV/TV and femoral BMD in the rats. These results illustrate that PBH-Ca has promising potential in a high-quality calcium diet.

The Role of Inhibitors in Ascertaining the Type of Isoenzyme of Alkaline Phosphatase (ALP) and its Clinical Correlations

Aims: To estimate the level of Alkaline Phosphatase (ALP) in the serum and to evaluate the role of inhibitors in ascertaining the type of isoenzyme of ALP and further correlation with clinical diagnosis and other biochemical parameters of the patients.&#x0D; Study Design: Cross Sectional with Diagnostic Accuracy Type&#x0D; Place and Duration of Study: Department of Biochemistry, Guru Gobind Singh Medical College, Faridkot, October 2017 to October 2018.&#x0D; Methdology: 250 Patients (143 women and 107 men; age range 19-85 years) with elevated ALP level were enrolled. ALP activity was measured in the serum and isoenzymes were ascertained by using chemical inhibitors i.e. Urea, L-Phenylalanine, Guanidine Hydrochloride, and heat inactivation which inhibit ALP activity. For heat inactivation, serum was incubated at 65°C for 10 minutes. Various other biochemical parameters were estimated for the correlation.&#x0D; Results: The Mean±SD of ALP level was 373±274 U/L. The percentage of inhibition by Urea, L-Phenylalanine, Guanidine hydrochloride and heat inactivation was 49.5%, 40.5%, 48.6%, and 94.0% respectively in liver disorders, 56.6%, 38.1%, 45.8%, and 89.0% in bone disorders and 29%, 41.6%, 42.8% and 95% in intestinal disorders. The percentage of inhibition in third-trimester pregnant females was 44.4%, 36.8%, 47.1%, and 36.8%, and 51.1%, 47.0%, 50.7%, and 51.2% in preeclampsia. The percentage of inhibition of the Isoenzyme of ALP was according to the type of carcinoma/lymphoma. The correlation of ascertained ALP isoenzyme with AST and ALT is highly significant (P&lt;0.001) in patients of liver diseases. In the patients of Bone disorders the correlation of ALP isoenzyme with Calcium and Phosphorous is also significant (P=0.01 and P=0.01 respectively).&#x0D; Conclusion: The sensitivity and specificity of this inhibition method to ascertain isoenzymes in raised ALP level was 82.45% and 66.13% respectively. The area under the ROC curve (AUC) is 0.744 which is significant (P&lt;0.0001).

Polypeptides IGF-1C and P24 synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the p38 and JNK signaling pathways.

Insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 2 (BMP-2) both promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs). IGF-1C, the C domain peptide of IGF-1, and P24, a BMP-2-derived peptide, both have similar biological activities as their parent growth factors. This study aimed to investigate the effects and mechanisms of polypeptides IGF-1C and P24 on the osteogenic differentiation of BMSCs. The optimum concentrations of IGF-IC and P24 were explored. The effects of the two polypeptides on BMSC proliferation and osteogenic differentiation were examined using a CCK-8 assay, flow cytometry, alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qPCR, and Western blotting. In addition, specific pathway inhibitors were utilized to explore whether the p38 and JNK pathways were involved in this process. The optimal concentration of both polypeptides was 50μg/ml. IGF-1C and P24 synergistically promoted BMSC proliferation, increased ALP activity and calcified nodule formation, upregulated the mRNA and protein levels of Osx, Runx2, Ocn, Opn, and Col1a1, and improved the phosphorylation levels of p38 and JNK proteins. Inhibition of the pathways significantly reduced p38 and JNK activation and blocked Runx2 expression while inhibiting ALP activity and calcified nodule formation. These findings suggest that IGF-1C and P24 synergistically promote the osteogenesis of BMSCs through activation of the p38 and JNK signaling pathways.

Pathological and Biochemical Index Changes in Different Tissues of Cyprinus carpio Exposed to Cadmium

Background: Cadmium (Cd) is among the most toxic metals and is widely distributed in aquatic environments. Objective: Histopathological lesions and biochemical indices can be used to reflect the toxic effects of cadmium in fish target organs. Methods: In the present study, the common carp Cyprinus carpio was exposed to 0.5 mg/L of CdCl2 for 21 days. Histopathological analyses were performed in the spleen, liver, kidney, gills and head kidney tissues of C. carpio, and tissue sections were stained with hematoxylin-eosin (HE) and observed under an optical microscope. The acid phosphatase, alkaline phosphatase, and lysozyme activities were determined. Results: The results showed that there were various pathological changes in five major tissues of C. carpio. The kidney, gills, and liver were the most affected organs during cadmium exposure, and deformation and disorganization of epithelial cells, vacuolization formation, serious hemorrhages and necrosis were also observed. In addition, 0.5 mg/L cadmium exposure significantly increased serum lysozyme and acid phosphatase activities and inhibited alkaline phosphatase activity. The present study showed that cadmium stress causes significant pathological tissue changes and influences lysozyme, acid phosphatase and alkaline phosphatase activities in C. carpio. Acid phosphatase and lysozyme activities are sensitive to cadmium. Conclusion: In conclusion, the toxicity of cadmium caused varying degrees of pathological and biochemical changes in fish tissues in C. carpio.

Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.

Sensitivity of midgut physiological parameters of Lymantria dispar L. larvae to benzo[a]pyrene in populations with different multigeneration contact to environmental pollutants

Accumulation of organic pollutants in the environment calls for sensing physiological parameters adequate to indicate the presence of contaminants and their effects on ecosystems. Evidence points to the importance of insect adaptations in their habitats for the assessment of sensitive biomarkers so we examined the influence of origin and multigenerational adaptations of the Lymantria dispar larvae to chronic benzo[a]pyrene (B[a]P) treatment under laboratory conditions. The main aim was to compare reactions of larvae from unpolluted and polluted forests using alkaline phosphatase (ALP), acid phosphatase (ACP), and carboxylesterase (CE) specific activities in the midgut, including electrophoretic isoform patterns; midgut expression levels of Hsp70, larval development time (DT), and midgut mass (MM), after chronic exposure to 5 and 50 ng of B[a]P/g dry food weight. The biomarker potential of these parameters regarding larval pre-exposure history to pollution was estimated by principal component analysis (PCA). B[a]P treatment resulted in inhibition of ALP activity, a rise of CE activity, and reduction of MM in larvae from the unpolluted forest, while the population from the polluted forest showed significant elevation of Hsp70 expression in the midgut, prolonged DT, and reduction of MM. PCA confirmed variations in responses of the selected parameters regarding population origin. The obtained results provide insight into insect population variability concerning physiological responses to pollutants. It is indicative that all investigated physiological parameters of L. dispar larvae showed origin-dependent responses to long-term presence of B[a]P, which may be of great importance in ecotoxicological research.

MicroRNA-383-5p regulates osteogenic differentiation of human periodontal ligament stem cells by targeting histone deacetylase 9

ObjectiveHuman periodontal ligament stem cells (hPDLSCs) play an important role in regenerative engineering technology for periodontal therapy. The mechanism of microRNA (miR)-383-5p in osteogenic differentiation needs further exploration. This study aimed at investigating the potential role of miR-383-5p in the osteogenic differentiation of hPDLSCs. MethodsOsteogenic differentiation of hPDLSCs was induced by osteoblastinducing media and evaluated by Alizarin Red staining and Alkaline phosphatase staining. To examine the role of miR-383-5p in osteogenic differentiation, miR-383-5p mimic or inhibitor and histone deacetylase (HDAC) 9 overexpression plasmid or siRNA-HDAC9 were co-transfected into hPDLSCs. qRT-PCR and Western blot were applied for detection of mRNA and protein levels. ResultsDuring the osteogenic differentiation of hPDLSCs, miR-383-5p expression was gradually up-regulated, while HDAC9 mRNA level was down-regulated. HDAC9 overexpression suppressed Alkaline phosphatase activity, mineral node formation and the expressions of osteogenic markers including Runx family transcription factor 2 (RUNX2), osteocalcin and Smad family member 4 (Smad4) in the differentiated hPDLSCs, while siHDAC9 exerted opposite effects on osteogenic differentiation. The Alkaline phosphatase activity, mineral node formation and the expressions of RUNX2, osteocalcin and Smad4 of the differentiated hPDLSCs were regulated by miR-383-5p/HDAC9 axis. The miR-383-5p/HDAC9 axis effectively regulated the expressions of osteogenic markers during the differentiation of hPDLSCs. ConclusionMiR-383-5p overexpression facilitated the osteogenic differentiation of hPDLSCs via inhibiting HDAC9 expression.

Biological indicators for environmental quality monitoring of marine sediment in Suez Gulf, Egypt

A comparative field study was performed to assess the heavy metals contamination in surficial sediments of highly and less polluted zones at the northern part of the Suez Gulf. In addition, to highlight the relationship among metal contamination and enzyme activity. Seven heavy metals Mn, Fe, Pb, Cd, Zn, Ni, and Cu were analyzed using atomic absorption spectrophotometer. The activity of enzymes (alkaline phosphatase and urease) was estimated in the sediment samples. The statistical results showed that there were high significant differences at p ≤ 0.01 among Cu, Pb, Cd, Ni, Mn and Zn between highly polluted and less polluted sediments. Urease activity had no significant relationship with the tested metals concentrations. Cd, Cu, Mn, Ni, Pb and Zn may inhibit alkaline phosphatase activity. Alkaline phosphatase activity showed high significant difference at p ≤ 0.01 between summer and winter seasons in highly polluted sediment. Moreover, there was significant seasonal difference in Pb concentration at p ≤ 0.01 in the same category. Correlations of the enzymes with each metal showed that alkaline phosphatase activity had significant negative correlation coefficients (r = −0.55, −0.49, −0.27, −0.37, −0.37, and −0.60) with Cu, Cd, Fe, Ni, Zn and Pb respectively in winter, but only with Zn (r = −0.46) in summer season. As a result, ALP activity can possibly be used as a predictor and sensitive indicator of heavy metals bioavailability in marine sediments.