Causal relationship between systemic circulatory inflammatory regulators and intervertebral disc degeneration: A bidirectional 2-sample Mendelian randomization study.

In the context of the development of intervertebral disc degeneration (IDD), inflammatory mediators play a pivotal role. Nevertheless, due to the influence of the inflammatory microenvironment, the causal relationship between specific inflammatory mediators and the development of IDD remains uncertain. The understanding of the causal relationship between inflammatory mediators and IDD is of great importance in preventing and delaying disc degeneration in the future. We utilized genetic data concerning systemic circulating inflammatory regulators obtained from a Genome-Wide Association Study (GWAS) analyzing 41 serum cytokines in a cohort of 8293 individuals from Finland. The genetic data for IDD were derived from the most recent GWAS summary statistics conducted within the FinnGen consortium, encompassing 37,636 IDD cases and 270,964 controls. Our analysis employed bidirectional 2-sample Mendelian randomization (MR) techniques, which included several MR methods such as MR Egger, weighted median, inverse variance weighted, weighted mode, and simple mode. Additionally, the MR-PRESSO method was employed to identify horizontal pleiotropy, heterogeneity was quantified using the Cochran Q statistic, and MR-Egger intercept analysis was performed to assess pleiotropy. We established causal relationships between 3 specific inflammatory factors and IDD. Elevated levels of MIP-1ß (OR = 0.956, 95% CI: -0.08 to -0.006; P = .02) and IFN-G (OR = 0.915, 95% CI: -0.16 to -0.02; P = .01) expression were associated with a reduced risk of IDD. Conversely, genetic susceptibility to IDD was linked to a decrease in IL-13 levels (OR = 0.967, 95% CI: -0.063 to -0.004; P = .03). In this study, we have identified inflammatory factors that exhibit a causal relationship with the onset and progression of IDD, as supported by genetic predictions.

METTL3 mediated ferroptosis in chondrocytes and promoted pain in KOA via HMGB1 m6A modification.

Methyltransferase-like 3 (METTL3) plays a role in the development of knee osteoarthritis (KOA). However, the mechanism underlying the role of METTL3 in KOA is unclear. This work investigated the effects of MELLT3 on ferroptosis and pain relief in in vitro and in vivo KOA models. Chondrocytes were treated with 10 ng/mL interleukin-1ß (IL-1ß) or 5 µM Erastin (ferroptosis inducer). IL-1ß or Erastin treatment inhibited cell viability and glutathione levels; increased Fe2+, lipid reactive oxygen species and malondialdehyde production; and decreased glutathione peroxidase 4, ferritin light chain and solute carrier family 7 member 11 levels. The overexpression of METTL3 facilitated the N6-methyladenosine methylation of high mobility group box 1 (HMGB1). HMGB1 overexpression reversed the effect of sh-METTL3 on IL-1ß-treated chondrocytes. A KOA rat model was established by the injection of monosodium iodoacetate into the joints and successful model establishment was confirmed by haematoxylin and eosin staining and Safranin O/Fast Green staining. METTL3 depletion alleviated cartilage damage, the inflammatory response, ferroptosis and knee pain in KOA model rats, and these effects were reversed by the addition of HMGB1. In conclusion, METTL3 depletion inhibited ferroptosis and the inflammatory response, and ameliorated cartilage damage and knee pain during KOA progression by regulating HMGB1.

Identification and experimental verification of immune-related hub genes in intervertebral disc degeneration.

Background: Inflammation and immune factors are the core of intervertebral disc degeneration (IDD), but the immune environment and epigenetic regulation process of IDD remain unclear. This study aims to identify immune-related diagnostic candidate genes for IDD, and search for potential pathogenesis and therapeutic targets for IDD. Methods: Gene expression datasets were obtained from the Gene Expression Omnibus (GEO). Differential expression immune genes (Imm-DEGs) were identified through weighted gene correlation network analysis (WGCNA) and linear models for microarray data analysis (Limma). LASSO algorithm was used to identify feature genes related to IDD, which were compared with core node genes in PPI network to obtain hub genes. Based on the coefficients of hub genes, a risk model was constructed, and the diagnostic value of hub genes was further evaluated through receiver operating characteristic (ROC) analysis. Xcell, an immunocyte analysis tool, was used to estimate the infiltration of immune cells. Finally, nucleus pulposus cells were co-cultured with macrophages to create an M1 macrophage immune inflammatory environment, and the changes of hub genes were verified. Results: Combined with the results of WGCNA and Limma gene differential analysis, a total of 30 Imm-DEGs were identified. Imm-DEGs enriched in multiple pathways related to immunity and inflammation. LASSO algorithm identified 10 feature genes from Imm-DEGs that significantly affected IDD, and after comparison with core node genes in the PPI network of Imm-DEGs, 6 hub genes (NR1H3, SORT1, PTGDS, AGT, IRF1, TGFB2) were determined. Results of ROC curves and external dataset validation showed that the risk model constructed with the 6 hub genes had high diagnostic value for IDD. Immunocyte infiltration analysis showed the presence of various dysregulated immune cells in the degenerative nucleus pulposus tissue. In vitro experimental results showed that the gene expression of NR1H3, SORT1, PTGDS, IRF1, and TGFB2 in nucleus pulposus cells in the immune inflammatory environment was up-regulated, but the change of AGT was not significant. Conclusions: The hub genes NR1H3, SORT1, PTGDS, IRF1, and TGFB2 can be used as immunorelated biomarkers for IDD, and may be potential targets for immune regulation therapy for IDD.

Zhiqiao Gancao decoction regulated JAK2/STAT3/ macrophage M1 polarization to ameliorate intervertebral disc degeneration.

Background: Zhiqiao Gancao decoction (ZQGCD) was created by Professor Gong Zhengfeng, a renowned Chinese medicine expert. Clinical studies have shown its efficacy in alleviating pain and enhancing lumbar function in intervertebral disc degeneration (IDD) patients. However, the precise mechanism of ZQGCD in treating IDD remains unclear. Methods: The active components of ZQGCD were identified using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A rat model of intervertebral disc degeneration was established, and rats in each group received ZQGCD for three weeks. Assessment parameters included hyperalgesia status, observation of intervertebral disc tissue degeneration and macrophage infiltration, and analysis of JAK2/STAT3 pathway protein expression in the intervertebral disc. Primary macrophage M1 polarization was induced using LPS, with cells treated using the JAK2 inhibitor (AZD1480) and ZQGCD to evaluate macrophage polarization, cellular supernatant inflammatory factors, and JAK2/STAT3 pathway expression. Macrophage supernatant served as a conditioned medium to observe its effects on the proliferation of nucleus pulposus cells (NPCs) and the expression of collagen II and MMP3 proteins. Results: A total of 81 active components were identified in ZQGCD. Following ZQGCD treatment, infiltrating macrophages in intervertebral disc tissues of model rats decreased, the content of M1 macrophages decreased, while the content of M2 macrophages increased, the expression of proinflammatory factors and pain-inducing factors in serum decreased, and the expression of substance P in intervertebral disc tissue decreased. Consequently, the intervertebral disc degeneration and hyperalgesia of rats were improved. In vitro studies revealed that LPS induced M1 macrophage polarization. By inhibiting the JAK2/STAT3 pathway, both JAK2 inhibitors and ZQGCD effectively suppressed M1 polarization, resulting in decreased levels of IL-1ß, IL-6, TNF-α, and various other inflammatory factors. Consequently, this inhibition led to a delay in the degeneration of NPCs. Conclusion: There is macrophage infiltration in the intervertebral disc tissue of IDD rats, and JAK2/STAT3 pathway is activated, macrophages are polarized to M1 type, resulting in inflammatory microenvironment, leading to intervertebral disc degeneration and hyperalgesia. ZQGCD exhibited a delaying effect on IDD and improved hyperalgesia by inhibiting the JAK2/STAT3/macrophage M1 polarization pathway.

An improved composite ship magnetic field model with ellipsoid and magnetic dipole arrays.

In order to simultaneously maintain the ship magnetic field modeling accuracy, reduce the number of coefficient matrix conditions and the model computational complexity, an improved composite model is designed by introducing the magnetic dipole array model with a single-axis magnetic moment on the basis of the hybrid ellipsoid and magnetic dipole array model. First, the improved composite model of the ship's magnetic field is established based on the magnetic dipole array model with 3-axis magnetic moment, the magnetic dipole array model with only x-axis magnetic moment, and the ellipsoid model. Secondly, the set of equations for calculating the magnetic moments of the composite model is established, and for the problem of solving the pathological set of equations, the least-squares estimation, stepwise regression method, Tikhonov, and truncated singular value decomposition regularization methods are introduced in terms of the magnetic field, and generalized cross-validation is used to solve the optimal regularization parameters. Finally, a ship model test is designed to compare and analyze the effectiveness of the composite and hybrid models in four aspects: the number of coefficient matrix conditions of the model equation set, the relative error of magnetic field fitting, the relative error of magnetic field extrapolation, and the computational time complexity. The modeling results based on the ship model test data show that the composite model can be used for modeling the magnetic field of ships, and compared with the hybrid model, it reduces the number of coefficient matrix conditions and improves the computational efficiency on the basis of retaining a higher modeling accuracy, and it can be effectively applied in related scientific research and engineering.

Zhiqiao Gancao Decoction Ameliorates Hyperalgesia in Lumbar Disc Herniation via the CCL2/CCR2 Signaling Pathway.

Purpose: The aim of this study was to investigate the effect of Zhiqiao Gancao decoction (ZQGCD) on hyperalgesia in lumbar disc herniation (LDH) and its mechanism. Methods: The potential mechanism of ZQGCD's therapeutic effect on LDH was investigated through network pharmacology, which involved screening the targets of eight components that were absorbed into the bloodstream. The effects of CCR2 inhibitors and ZQGCD-containing serum on the excitability of the CCL2/CCR2 signaling pathway and dorsal root ganglion neurons (DRGn) were investigated in vitro. The effects of CCR2 inhibitors and ZQGCD on the expression of the CCL2/CCR2 signaling pathway and ASIC3 in the rat intervertebral disc and dorsal root ganglion (DRG), the degree of disc degeneration, the threshold of foot retreat, and the latency of foot retreat in LDH rats were examined in vivo. The binding affinities and interaction modes between CCR2 and the components absorbed into the blood were analyzed using the AutodockVina 1.2.2 software. Results: Network pharmacology revealed that ZQGCD could treat LDH through a mechanism involving the chemokine signaling pathway. It was observed that the CCR2 inhibitor and ZQGCD-containing serum downregulated CCR2 and ASIC3 expression and decreased cell excitability in DRGn. The CCL2/CCR2 signaling pathway was activated in the degenerated intervertebral disc and DRG of LDH rats, increased the expression of ASIC3, and decreased the mechanical allodynia domain and thermal hyperalgesia domain. However, a CCR2 inhibitor or ZQGCD could ameliorate the above changes in LDH rats. The target proteins, CCL2 and CCR2, exhibited a robust affinity for the eight components that were absorbed into the bloodstream. Conclusion: The CCL2/CCR2 pathway was activated in the intervertebral disc and DRG of LDH rats. This was accompanied by upregulation of ASIC3 expression, increased excitability of DRGn, and the occurrence of hyperalgesia. ZQGCD improves hyperalgesia in LDH rats by inhibiting the CCL2/CCR2 pathway and downregulating ASIC3 expression.

Demethoxycurcumin mitigates inflammatory responses in lumbar disc herniation via MAPK and NF-κB pathways in vivo and in vitro.

The inflammatory radicular pain induced by lumbar disc herniation (LDH) is a serious problem worldwide. Demethoxycurcumin (DMC) is a yellow pigment derived from turmeric. Although it is considered a safe natural compound for managing inflammation-associated diseases, but the molecular mechanisms of LDH remain to be elucidated. In the current study, DMC reduced the production of IL-1ß, IL-4, and IL-6 in nucleus pulposus (NP) cells subjected to TNF-α-induced inflammation. Moreover, the inhibitory mechanism was activated upon suppression of activation of MAPKs and NF-κB signalling in NP cells. Further experiments with LDH model rats supported the in vitro results. These studies expand our knowledge of the effect of DMC on LDH; DMC may be a viable alternative to the drugs used to treat LDH.

Cross-iteration multi-step optimization strategy for three-dimensional intensity position correction in phase diverse phase retrieval.

Parameters mismatching between the real optical system and phase retrieval model undermines wavefront reconstruction accuracy. The three-dimensional intensity position is corrected in phase retrieval, which is traditionally separated from lateral position correction and axial position correction. In this paper, we propose a three-dimensional intensity position correction method for phase diverse phase retrieval with the cross-iteration nonlinear optimization strategy. The intensity position is optimized via the coarse optimization method at first, then the intensity position is cross-optimized in the iterative wavefront reconstruction process with the exact optimization method. The analytic gradients about the three-dimensional intensity position are derived. The cross-iteration optimization strategy avoids the interference between the incomplete position correction and wavefront reconstruction during the iterative process. The accuracy and robustness of the proposed method are verified both numerically and experimentally. The proposed method achieves robust and accurate intensity position correction and wavefront reconstruction, which is available for wavefront measurement and phase imaging.

Demethoxycucumin protects MDA-MB-231 cells induced bone destruction through JNK and ERK pathways inhibition.

Bone is the most common late metastasis of breast cancer. Bone metastasis causes not only severe bone pain, but also bone-related diseases such as pathological fractures, which are closely related to osteoclasts. The effects of demethoxycurcumin (DMC) on osteoclast biology has not been investigated. In this study, we explored the effects of DMC on MDA-MB-231 cells, MCF-7 cells, and osteoclasts induced by RANKL in vitro, as well as the protective effect on bone destruction of tumor bone metastasis in vivo. DMC showed inhibitory effect on the migration and promotes the apoptosis of MDA-MB-231 and MCF-7 cells. At the same time, DMC inhibited osteoclast maturation and mature osteoclast bone resorption in a dose-dependent manner, and suppressed the expression of osteoclast marker genes TRAP, CTSK, MMP9, V-ATPase-d2 and DC-STAMP significantly. Biochemical data showed that DMC inhibited tumor cells and osteoclasts by inhibiting the early activation of ERK and JNK MAPK pathway. Consistent with the results in vitro, we confirmed that DMC protects bone destruction caused by tumor metastasis in vivo. In short, our study confirmed that DMC could be used as a potential drug for the treatment of tumor bone destruction.

Modal-based nonlinear optimization algorithm for wavefront measurement with under-sampled data.

The aliasing effect in the discrete Fourier transform inherent will impose a serious detrimental effect on conventional phase retrieval measurement accuracy with under-sampled intensity. In this Letter, we describe a modal-based nonlinear optimization phase retrieval approach that is capable of retrieving wavefront measurements using under-sampled intensities. The extended Nijboer-Zernike theory is introduced to establish an analytic solution between wavefront phase and intensity image, and then nonlinear optimization is further utilized to solve wavefront aberration coefficients from under-sampled intensity data. The feasibility and accuracy of the algorithm are verified by simulations and experiments. This is a promising method that is especially suitable for full field phase recovery of optical systems with a relatively high numerical aperture.

Icariin controlled release on a silk fibroin/mesoporous bioactive glass nanoparticles scaffold for promoting stem cell osteogenic differentiation.

The treatment of bone defects caused by various reasons is still a major problem in orthopedic clinical work. Many studies on osteogenic implant materials have used various biologically active factors such as osteogenic inducers, but these biologically active factors have various side effects. Therefore, in this study, silk fibroin (SF) was used as a scaffold material, mesoporous bioactive glass nanoparticles (MBGNs) as a sustained release carrier, and the traditional Chinese drug icariin (ICA) was loaded to promote bone formation. The experiments in this study have proven that SF/MBGNs-ICA scaffolds can successfully load and release ICA for a long time, and the sustained-release ICA can promote the proliferation and differentiation of BMSCs for a long time. This controlled-release ICA organic/inorganic two-component scaffold material is expected to become a new bone grafting solution.

[Treatment of anterior glenohumeral dislocations with manual reduction of WU medical school].

OBJECTIVE: To investigate the technique, mechanism and clinical efficacy of manual reduction of WU medical school in the treatment of anterior glenohumeral dislocations. METHODS: From January 2016 to December 2017, 181 patients with anterior glenohumeral dislocations were treated with our manual reduction, including 71 males and 110 females, ranging in age from 19 to 94 years old, with a mean age of(61.1±16.3) years old; 68 cases of subglenoid type, 93 cases of subcoracoid type and 20 cases of subclavian type. Constant score was used to evaluate limb function while the external fixation was removed. RESULTS: One hundred and fifty-seven patients achieved reduction at the first attempt and 23 patients achieved at the second time. There was no vascular damage, nerve damage or iatrogenic fracture accmpanied. The Constant score ranged from 75 to 100, with a mean score of 92.1±4.3. One hundred and sixty-eight patients were followed up, and the duration ranged from 12 to 24 months, with an average of (16.1±3.2) months, no recurrent dislocation occurred during the follow up period. CONCLUSIONS: The manual reduction of WU medical school in the treatment of anterior glenohumeral dislocations has high success rate and low complication rate, which is scientific, safe, standardized, easy to learn and worth promoting.

[Treatment of thoracolumbar fractures with three-dimensional reduction method and external fixation devices under analgesia].

OBJECTIVE: To compare the clinical effects between open reduction internal fixation and three-dimensional reduction with external fixation under analgesia in treating fresh thoracolumbar fractures, and explore the simple and effective method for thoracolumbar fractures. METHODS: The clinical data of 40 patients with thoracolumbar fractures who met the inclusion and exclusion criteria in the department of orthopaedics affiliated to Suzhou Hospital of Nanjing University of Chinese Medicine from February 2013 to August 2017 were retrospectively analyzed. According to therapeutic methods, the patients were devided into treatment group and control group, 20 cases in each group. Treatment group was treated by three-dimensional reduction method and external fixation devices under analgesia, and control group was treated by open reduction and common spinal fixation system. In treatment group, there were 9 males and 11 females, aged from 26 to 68 years old with an average of (52.8±11.3) years; and in control group, there were 10 males and 10 females, aged from 26 to 64 years old with an average of(50.6±8.8) years. Anterior vertebral body compression(AVBC), Cobb angle and visual analogue scale(VAS) were measured and compared in two group. RESULTS: All 40 patients finished follow-up. The follow-up time in treatment group was 5 to 37 months with average of (16.1±8.8) months, in control group was 5 to 29 months with an average of(17.3±6.0) months. There was no significant difference between two groups(P>0.05). AVBC, Cobb angle, VAS score were obviously improved in all patients after treatment(P<0.05), but there were no significant difference between two groups(P>0.05). CONCLUSIONS: Clinical effect of two methods was similar in treating thoracolumbar fractures, but three-dimensional reduction and external fixation devices under analgesia has advantage of easy operation, smaller trauma and no need secondary surgery for removed internal fixation.

Identification, characterization and expression analysis of MAVS in Pelodiscus sinensis after challenge with Poly I:C.

Pelodiscus sinensis, which is one of the important reptile species in the aquaculture industry in China, frequently suffers from serious infectious diseases caused by viruses. However, there is a lack of biological knowledge about its antiviral innate immunity. In this study, we identified and characterized the open reading frame (ORF) of PsMAVS cDNA in P. sinensis. It consisted of 2691 nucleotides encoding a protein of 896 amino acid residues, which were composed of an N-terminal CARD, a central proline-rich domain and a C-terminal TM domain. Based on the amino acid sequence, phylogenetic analyses revealed a closer relationship of PsMAVS with those of Chelonia. qRT-PCR analysis indicated that PsMAVS was ubiquitously expressed in all of the examined healthy tissues with different expression levels; it was expressed at high levels in spleen, muscle and heart and at moderate levels in kidney, liver, intestine, intestinum crissum and oesophagus. PsMAVS was detected in embryos at 10 days post hatching, and it gradually upregulated with the embryonic development stage. Its expression levels in the examined tissues were all upregulated significantly after challenge with Poly I:C. The PsMAVS protein was detected in the intestinal tissues from both the challenge and the control groups, and it was distributed widely in the cytoplasm of the intestinal cells, suggesting PsMAVS plays multiple roles in the complicated mechanisms of immune defence against virus invasion in P. sinensis.

[Percutaneous poking reduction with bone grafting and limited internal fixation for the treatment of calcaneal fractures].

OBJECTIVE: To investigate clinical effects of percutaneous poking reduction with bone grafting and limited internal fixation for the treatment of calcaneal fractures. METHODS: From May 2013 to October 2016, 53 patients with closed calcaneal fractures were analyzed, and were divided into treatment group and control group. There were 33 patients in treatment group including 25 males and 8 females, aged from 15 to 82 years old with an average of(44.7±14.2) years old; 17 cases were type II and 16 cases were type III according to Sanders classification; treated by percutaneous poking reduction with bone grafting and limited internal fixation. There were 33 patients in control group, including 20 males aged from 25 to 62 years old with an average of (42.2±11.3) years old; 8 cases were type II and 12 cases were type III according to Sanders classification; treated by open reduction and internal fixation. Imaging indicators, hospital stays and preoperative waiting time were observed and compared, Maryland scoring were applied to evaluate foot function. RESULTS: Fifty-three patients were followed up, and treatment group was followed up from 8 to 40 months with an average of (19.9±7.2) months; while control group was followed up from 12 to 40 months with an average of (21.7±7.7) months, and there were not significant differences between two groups in follow-up time (P>0.05). There were no obvious meaning in Böhler angles, Gissane angles between two groups (P>0.05). There were significant differences in hospital stays and preoperative waiting time(P<0.01). There were no significant differences in Maryland score between treatment group(90.45±5.76) and control group(89.10±6.16). CONCLUSIONS: Percutaneous poking reduction with bone grafting and limited internal fixation for the treatment of calcaneal fractures could obtain satisfied effects, and has advantages of less trauma and complications, rapid recovery and good clinical effects.

Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis.

Pelodiscus sinensis is the most common turtle species that has been raised in East and Southeast Asia. However, there are still limited studies about the immune defense mechanisms in its small intestine until now. In the present research, histological analysis and transcriptome analysis was performed on the small intestine of P. sinensis after intragastric challenge with LPS to explore its mechanisms of immune responses to pathogens. The result showed the number of intraepithelial lymphocytes (IELs) and goblet cells (GCs) in its intestine increased significantly at 48 h post-challenge with LPS by intragastrical route, indicating clearly the intestinal immune response was induced. Compared with the control, a total of 748 differentially expressed genes (DEGs) were identified, including 361 up-regulated genes and 387 down-regulated genes. Based on the Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG), 48 immune-related DEGs were identified, which were classified into 82 GO terms and 14 pathways. Finally, 18 DEGs, which were randomly selected, were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide valuable information for further analysis of the immune defense mechanisms against pathogens in the small intestine of P. sinensis.

Identification, characterization and expression analysis of ERK2 in Chinese mitten crab Eriocheir sinensis after challenge with LPS and Aeromonas hydrophila.

Farming of Eriocheir sinensis was seriously threaten by the infection of opportunistic pathogens, especially the gram-negative bacterium. In this paper, we analyzed the sequence of extracellular signal-regulated kinases 2 (ERK2) of E. sinensis (EsERK2) and its expression levels after challenge with LPS and Aeromonas hydrophila in both in vivo and in vitro examination. The full-length cDNA sequence of EsERK2 was 2455 bp in size with an open reading frame (ORF) of 1095 bp, encoding the protein of 365 amino acids. It owned a predicted molecular mass of 42.4 kDa and a theoretical isoelectric point (pI) of 5.93. EsERK2 was distributed in all examined tissues including haemocyte, gonad, hepatopancreas, gill, muscle heart, stomach and intestine, but its expression level was significantly higher in hepatopancreas than it in other examined tissues. The expression level of EsERK2 increased significantly after LPS challenge at 2 h (P < 0.05), and then gradually increased and reached highest at 16 h. However, its expression level decreased significantly after A. hydrophila challenge at 4 h, and then gradually decreased till 24 h (P < 0.05), and returned to its initial value at 36 h. According to the immunofluorescence assay and western blotting assay, EsERK2 was found to be distributed mainly in cytoplasm of haemocyte, and its expression level showed a prominent boost in primary cultured haemocytes after challenge with LPS and A. hydrophila in vitro. These results indicated that the expression of EsERK2 was sensitive to the exterior stimulants and its encoding protein might be associated with the signaling transduction in response to exterior pathogens in E. sinensis.

Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis.

In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 µg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.

Immune response of peroxinectin of Chinese mitten crab Eriocheir sinensis to exterior stimulation.

Peroxinectin possesses the features of both peroxidase activity and adhesive property and plays important roles in innate immune system of crustaceans. In this study, the sequence of peroxinectin of Eriocheir sinensis (EsPX) was analyzed and its expression in response to exterior stimulation was detected in both in vivo and in vitro examination. We showed that the full-length cDNA sequence was composed of 2701 bp and owned a molecular mass of 85.2 kDa and a theoretical pI (isoelectric point) of 6.91. Real-time PCR revealed that the EsPX was mainly distributed in the muscle, hemocytes and stomach. Furthermore, the EsPX was verified to be located in hyalinocytes, semigranulocytes and granulocytes, and was distributed throughout the cytoplasm and nucleus, especial in cytoplasm. After injected with beads, lipopolysaccharide (LPS) and Aeromonas hydrophila, the EsPX mRNA expression was significantly up-regulated and peaked up at 4, 2 and 16 h respectively (P <0.05). In the in vitro experiment, the stimulation of LPS and beads also induced a prominent boost of EsPX protein in primary cultured hemocytes. The expression of EsPX was peaked up at 4 and 8 h for LPS and beads challenged groups respectively, followed by remarkable release of the incremental EsPX into the extracellular matrix. These findings suggested that the expression of EsPX was susceptible to exterior stimulation, and that the highly expressional EsPX would be released into extracellular matrix by the exterior stimulus.

Effect of a new drug releasing system on microencapsulated islet transplantation.

OBJECTIVE: This study aimed to develop a novel release system for grafted islets. MATERIALS AND METHODS: A graphene oxide-FTY720 release system was constructed to test the drug loading and releasing capacity. The recipient rats were divided into four groups as following: Experiment group A (EG A) and B (EG B); Control group A (CG A) and B (CG B). In each group, (2000 ± 100) IEQ microencapsulated islets were implanted into the abdominal cavity of the recipients with oral FTY720, local graphene oxide-FTY720 injection, without immunosuppressants, and with graphene oxide-saturated solution respectively. We detected the immunological data, the blood glucose level, and pericapsular overgrowth to show the transplantation effect. RESULTS: 31% of adsorptive FTY720 was released within 6 h, and 82% of FTY720 was released within 48 h. From day 5 to 8, the amount of PBL in EG B was significantly less than those in EG A (P<0.01). The CD3+ and CD8+ T lymphocytes were suppressed 3 days longer in EG B than in EG A. On day 19 posttransplantation, the blood glucose level in EG B was much lower than that in EG A (P<0.01). On the same day, pericapsular overgrowth was grade I in EG B, grade II in other groups. CONCLUSIONS: Graphene oxide-FTY720 complex showed a drug releasing effect. Local application of graphene-FTY720 releasing system could decrease the amount of peripheral blood lymphocytes (PBL) and the percentage of CD3 and CD8 T lymphocytes in blood for longer time than oral drug application. This releasing system could achieve a better blood glucose control.