Organophosphorus flame retardants and their metabolites in paired human blood and urine.

Organophosphorus flame retardants (OPFRs) have been shown to be carcinogenic, neurotoxic, and endocrine disruptive, so it is important to understand the levels of OPFRs in human body as well as the modes of external exposure. In this study, we investigated the levels of 13 OPFRs and 7 phosphodiester metabolites in paired human blood and urine, as well as the influencing factors (region, age and gender), and studied the relationship between OPFRs and oxidative stress by urinary metabolites. We found that the concentrations of triphenyl phosphate (TPhP) and tris-(2-ethylhexyl) phosphate (TEHP) in the blood of urban populations were higher than those of rural populations, and that younger populations suffered higher TPhP and 2-ethylhexyl diphenyl phosphate (EHDPP) exposures than older populations. In addition, we found that tris-(2-chloroethyl) phosphate (TCEP), tributyl phosphate (TnBP), TPhP and EHDPP exposure induced oxidative stress. The results of the internal load principal component analysis indicated that dust ingestion, skin exposure, respiration and dietary intake may be the most important sources of TCEP, tris(2-butoxyethyl) phosphate (TBOEP), tri(2-chloroisopropyl) phosphate (TCIPP) and TEHP, respectively, and dust ingestion and skin exposure may be the main sources of TPhP for humans.

[Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway].

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-ß-galactosidase(SA-ß-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1ß(IL-1ß), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1ß and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1ß and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Aucubin inhibits IL-1ß- or TNF-α-induced extracellular matrix degradation in nucleus pulposus cell through blocking the miR-140-5p/CREB1 axis.

In intervertebral disc degeneration (IDD), increased proinflammatory molecules secreted by human nucleus pulposus cells (HNPCs) could promote the expression of extracellular matrix (ECM)-degrading enzymes. IDD could be affected by both genetic and environmental factors, including microRNAs (miRNAs). Aucubin, the active ingredient of a traditional Chinese medicine herb Du Zhong, has been reported to promote osteogenic differentiation; however, the role of aucubin in IDD and the underlying mechanism remain unclear. Herein, we evaluated the effect of aucubin on TNF-α- or IL-1ß-induced ECM degradation in HNPCs. By using online tools, miR-140 was selected as a candidate miRNA that is related to TNF-α or IL-1ß signaling. Overexpression of miR-140 enhanced the effect of aucubin on ECM degradation. Moreover, cAMP responsive element binding protein 1 (CREB1), a major transcriptional factor in immune-related signaling, was a direct downstream target of miR-140. CREB1 knockdown mimicked the function of miR-140 overexpression on ECM degradation. In summary, aucubin might ameliorate IL-1ß- or TNF-α-induced ECM degradation in HNPCs through regulating miR-140/CREB1.

Bu-Shen-Huo-Xue-Fang modulates nucleus pulposus cell proliferation and extracellular matrix remodeling in intervertebral disk degeneration through miR-483 regulation of Wnt pathway.

Intervertebral disk degeneration (IDD) has been widely considered as one of the main causes for low back pain, which can cause a severe impact to human health and huge economic burden to worldwide society. IDD pathogenesis can be affected by extensive degradation of extracellular matrix (ECM) and the hyperproliferation of nucleus pulposus (NP) cells. During the IDD process, expression of the ECM degradation enzymes matrix metalloproteinase and ADAMTS increases, whereas expression of ECM synthesis-related aggrecan and COL2A1 decreases. In addition, the Wnt signaling pathway is reportedly involved in the process of IDD. Bu-Shen-Huo-Xue-Fang (BSHXF), a Chinese traditional medicine formula that contains six Chinese traditional medicinal herbs, is widely used in the treatment of IDD. Herein, we obtained the serum containing BSHXF from BSHXF-fed rat and demonstrated that the BSHXF promoted NP cell proliferation and ECM synthesis through the Wnt signaling pathway. By using DIANA online tools and luciferase reporter gene assays, we confirmed that miR-483-3p and miR-23c regulated CTNNB1 and GSK3B, respectively, through direct targeting, thereby affecting the effect of BSHXF on NP cell proliferation and ECM synthesis through the Wnt signaling pathway. Taken together, we demonstrated the function and mechanism of BSHXF in regulating NP cell proliferation and ECM remodeling through the Wnt signaling pathway during IDD.

Psoralidin Induced Differentiation from Adipose-derived Stem Cells to Nucleus Pulposus-like Cells by TGF-ß/Smad Signaling.

BACKGROUND: Psoralidin (PL) could affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). The role of PL is still unclear in adipose-derived stem cells (ADSCs). AIMS: This study aimed to investigate the effects of PL on ADSCs differentiation into nucleus pulposus-like cells and the TGF-ß/Smad signaling pathway. METHODS: The proliferation and apoptosis of ADSCs were detected. The nucleus pulposus cell-related markers (CD24, BASP1, KRT19, and Aggrecan) and TGF-ß/Smad signaling pathway indexes were analyzed. RESULTS: The results showed that compared to the control group, the cell activity was increased in the PL group, and the apoptosis rate was decreased. The mRNA and protein levels of nucleus pulposus cells markers (CD24, BASP1, KRT19, Aggrecan, and Collagen Type II) and TGF-ß/Smad signaling pathway-related indexes (TGF-ß, SMAD2, and SMAD3) were increased in PL group. After treatment with PL and TGF-ß silencing, the TGF-ß/Smad signaling pathway-related indicators (TGF-ß, SMAD2, and SMAD3) and nucleus pulposus cells markers (CD24, BASP1, KRT19, Aggrecan, and Collagen Type II) were found to be higher in the sh-TGF-ß +PL group than in the sh-TGF-ß group. CONCLUSION: In conclusion, our study showed that PL might induce the differentiation of ADSCs to nucleus pulposus cells through the TGF-ß/Smad signaling pathway. It might have the potential application value in the treatment of intervertebral disc degeneration.

Psoralen synergizes with exosome-loaded SPC25 to alleviate senescence of nucleus pulposus cells in intervertebral disc degeneration.

OBJECTIVE: To explore the mechanism of psoralen synergized with exosomes (exos)-loaded SPC25 on nucleus pulposus (NP) cell senescence in intervertebral disc degeneration (IVDD). METHODS: IVDD cellular models were established on NP cells by tert-butyl hydroperoxide (TBHP) induction, followed by the treatment of psoralen or/and exos from adipose-derived stem cells (ADSCs) transfected with SPC25 overexpression vector (ADSCs-oe-SPC25-Exos). The viability, cell cycle, apoptosis, and senescence of NP cells were examined, accompanied by the expression measurement of aggrecan, COL2A1, Bcl-2, Bax, CDK2, p16, and p21. RESULTS: After TBHP-induced NP cells were treated with psoralen or ADSCs-oe-SPC25-Exos, cell proliferation and the expression of aggrecan, COL2A1, Bcl-2, and CDK2 were promoted; however, the expression of Bax, p16, p21, and inflammatory factors was decreased, and cell senescence, cycle arrest, and apoptosis were inhibited. Of note, psoralen combined with ADSCs-oe-SPC25-Exos further decelerated NP cell senescence and cycle arrest compared to psoralen or ADSCs-oe-SPC25-Exos alone. CONCLUSION: Combined treatment of psoralen and ADSCs-oe-SPC25-Exos exerted an alleviating effect on NP cell senescence, which may provide an insightful idea for IVDD treatment.

Enhancing nitrogen removal in an Orbal oxidation ditch by optimization of oxygen supply: practice in a full-scale municipal wastewater treatment plant.

Seven different aeration modes, in which oxygen supply was changed by adjusting the number of aerators, were designed and applied in a full-scale municipal wastewater treatment plant with Orbal oxidation ditch to investigate the influence of dissolved oxygen (DO) on nitrogen removal performance. The full-scale experiment results of 574 days showed that nitrogen removal efficiency depended on the degree of nitrification and denitrification in the outer channel, which was the largest contributor for TN removal in the Orbal oxidation ditch. Appropriate aeration control in the outer channel was essential to balance nitrification and denitrification in the Orbal oxidation ditch. When DO was as low as about 0.2 mg/L in the outer channel, the highest TN removal efficiency of 75% was obtained. Microbial analysis confirmed that aerobic and anaerobic bacteria coexisted in the outer channel. The greater species diversity and more intensive activities of these bacteria in aeration Mode V may be responsible for the higher TN removal efficiency compared with Mode III. These results suggest that different aerated conditions in the Orbal oxidation ditch might have a significant effect on microbial community characteristics and nitrogen removal efficiencies.

Traditional Chinese medicine therapies for patients with knee osteoarthritis: A protocol for systematic review and network meta-analysis.

BACKGROUND: Knee osteoarthritis (KOA) is a common cause of chronic musculoskeletal pain and disability as well as a socioeconomic burden on healthcare services globally. Numerous clinical trials indicated that traditional Chinese medicine (TCM) may effectively improve the clinical symptoms of KOA patients. However, the comparative efficacy and safety of different TCM therapies in patients with KOA is not yet clear. In order to evaluate the efficacy and safety of TCM for KOA, we will conduct a systematic review and network meta-analysis on the existing randomized controlled trials (RCTs). METHODS: A systematic literature search will be conducted in PubMed, Web of Science, Embase, EBSCO, Cochrane Library, China National Knowledge Infrastructure, Wanfang, Chinese Biomedical Literature Database, and the VIP Database for Chinese Technical Periodicals up to February 2022 to identify the relevant RCTs. The primary outcomes are visual analog scale, Western Ontario and McMaster Universities Osteoarthritis Index, Lysholm score, and Lequesne index. Secondary outcomes include the total clinical effective rate and adverse events. Study quality will be evaluated using the Cochrane risk of bias tool (RoB 2.0) for RCTs. Data analysis will be performed using Stata and WinBUGS. The quality of evidence will be assessed using the Grades of Recommendations Assessment Development and Evaluation. RESULTS: The results of this study will be submitted to a peer-reviewed journal for publication. CONCLUSIONS: This study will provide evidence-based medical evidence for the treatment of KOA with TCM therapies and offer better assistance for clinical practice. PROTOCOL REGISTRATION NUMBER: INPLASY202230008.

Achyranthoside D (AD) improve intervertebral disc degeneration through affect the autophagy and the activation of PI3K/Akt/mTOR pathway.

PURPOSE: This study aims to explore the potential mechanism of Achyranthoside D (AD) in improving intervertebral disc (IVD) degeneration (IDD). METHODS: The IDD model of SD rats and nucleus pulposus cells (NPCs) was established by lumbar cone annulus puncture and tert-butyl peroxide, respectively. Cell proliferation was detected by CCK8 assay. Apoptosis was detected by flow cytometry and TUNEL staining. IVD tissue injury was observed by HE staining. Alcian blue staining observed the glycoprotein secretion in IVD. Monodansylcadaverin (MDC) staining was used to detect the formation of autophagosomes. The LC3 expression was tested by immunofluorescence. The type II collagen, aggrecan and MMP3 expression were detected by ELISA. RT-qPCR was used to detect the Casp 3, Bax, Bcl2, Acan, Col2a1 and Mmp3 expression. The LC3, P62, type II collagen, aggrecan, Beclin1, Akt, MMP3, p-mTOR, PI3K, mTOR, p-PI3K and p-Akt expression were analyzed by western blot. RESULTS: The IVD tissue damage and apoptosis occurred in the Model group, and the glycoprotein secretion decreased. Compared with Model group, AD-H group alleviated the injury of IVD tissue, inhibited the apoptosis of cells, and increased the secretion of glycoprotein. 40 µg/mL AD restored the proliferation activity of NPCs. Compared to the Normal group, the NPCs apoptosis increased, the Collagen II, aggrecan and Bcl2 expressions were significantly decreased, the MMP3, Bax and Casp 3 expression were significantly increased, and the LC-3 II/I expression in IVD tissues were increased significantly in Model group, all of which was reversed in AD group. AD promoted the p-Akt, p-PI3K, p-mTOR, LC-3 II/I and Beclin1 expression, inhibited the P62 expression to alleviate the damage of nucleus pulporeus cells and the degeneration of IVD. CONCLUSION: AD improved IDD by affecting the PI3K/Akt/mTOR pathway and autophagy.

Protein transduction domain can enhance the humoral immunity and cross-protection of HPV16L2 peptide vaccines.

Due to type-specificity, commercially available human papillomavirus (HPV) vaccines are only effective against homologous HPV serotypes, providing limited protection. Recent studies have highlighted the role of HPV minor capsid protein (known as L2) in inducing cross-protection. The N-terminal peptides of L2 contain conserved cross-response epitopes that can induce neutralizing antibodies against heterogeneous HPVs. However, when compared with L1, these peptides have lower immunogenicity, which limits the application of these vaccines. The protein transduction domain (PTD), located in the Tat protein of human immunodeficiency virus, facilitates delivery of DNA, peptides, proteins and virus particles into cells by unknown mechanisms, and has been reported to enhance immunogenicity of several antigens. In the present study, two peptides derived from the N-terminal of HPV16L2 were chosen as model antigens and constructed a series of L2 peptide vaccines by either fusing or mixing with PTD. Subsequently their immunogenicity was evaluated. The results indicated that the L2 peptides fused with PTD show considerably enhanced humoral immunity. In particular, they increased the titer of cross-neutralizing antibodies, while L2 peptides that had only been mixed with PTD induced only small cross-protection responses. Overall, the data suggest that fusion of L2 peptides with PTD significantly enhances their cross-protection and may be a promising strategy for the development of broad-spectrum HPV prophylactic vaccines.

[Effect of Jingang Jiangu pill (see text) on expression of integrin beta1 and alphavbeta3 in ovariectomized osteoporosis model rats].

OBJECTIVE: To investigate the regulatory effect of Jingang Jiangu pill (see text, JGJG) on expression of integrin in ovariectomized rats. METHODS: Fifty ovariectomized 10 months old female rats were randomly divided into 5 groups: Fushanmei group (FSM), Jingang Jiangu pill (see text) group (JGJG), Gusongbao granule group (GSB), Model group (OVX), Sham group. After ovariectomized,the rats were raised in the same environment for 13 weeks. The rats in JGJG group took 0.13 g JGJG pill orally each day for each rat; the rats in GSB group took 0.86 g GSB granule orally each day for each rat; the rats in FSM group took 0.28 mg FSM orally each day for each rat; and the rats in OVX and sham groups took sodium. The treatment duration of rats in above 5 groups was 13 weeks. Bone mineral density (BMD) and the expression of integrin beta1 and alphavbeta3 were detected in each group after the treatment. RESYKTS: The BMD and the expression of integrin beta1 in FSM group, JGJG group and GSB group improved obviously than that of OVX group. There were statistical difference between these groups (P<0.05). The expression of integrin alphavbeta3 of the three treating groups significantly depressed. CONCLUSION: The JGJG pill improves BMD and express of integrin beta1, in ovariectomized rats and reduces express of integrin alphavbeta3 through the regulation of the coupling of osteoblasts and osteoclasts.

[Chromatographic separation of plasmid DNA by anion-exchange cryogel].

Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

Microchannel liquid-flow focusing and cryo-polymerization preparation of supermacroporous cryogel beads for bioseparation.

Polymeric cryogels are sponge-like materials with supermacroporous structure, allowing them to be of interest as new chromatographic supports, cell scaffolds and drug carriers in biological and biomedical areas. The matrices of cryogels are always prepared in the form of monoliths by cryo-polymerization under frozen conditions. However, there are limited investigations on the production of cryogels in the form of adsorbent beads suitable for bioseparation. In this work, we provide a new approach by combining the microchannel liquid-flow focusing with cryo-polymerization for the preparation of polyacrylamide-based supermacroporous cryogel beads with a narrow particle size distribution. The present method was achieved by introducing the aqueous phase solution containing monomer, cross-linker and redox initiators, and the water-immiscible organic oil phase containing surfactant simultaneously into a microchannel with a cross-shaped junction, where the aqueous drops with uniform sizes were generated by the liquid shearing and the segmentation due to the steady flow focusing of the immiscible phase streams. These liquid drops were in situ suspended into the freezing bulk oil phase for cryo-polymerization and the cryogel matrix beads were obtained by thawing after the achievement of polymerization. By grafting the polymer chains containing sulfo binding groups onto these matrix beads, the cation-exchange cryogel beads for protein separation were produced. The results showed that at the aqueous phase velocities from 0.5 to 2.0 cm/s and the total velocities of the water-immiscible phase from 2.0 to 6.0 cm/s, the obtained cryogel beads by the present method have narrow size distributions with most of the bead diameters in the range from 800 to 1500 µm with supermacropores in sizes of about 3-50 µm. These beads also have high porosities with the averaged maximum porosity of 96.9% and the mean effective porosity of 86.2%, which are close to those of the polyacrylamide-based cryogel monoliths. The packed bed using the cryogel beads with mean diameter of 1248 µm, as an example, has reasonable and acceptable liquid dispersion, but high water permeability (4.29 × 10⁻¹° m²) and high bed voidage (90.2%) owing to the supermacropores within the beads, enhanced the rapid binding and separation of protein from the feedstock even at high flow velocities. The purity of the obtained lysozyme from chicken egg white by one-step chromatography using the packed bed was in the range of about 78-92% at the flow velocities of 0.5-15 cm/min, indicating that the present cryogel beads could be an effective chromatographic adsorbent for primary bioseparation.