Influence of soil depth, irrigation, and plant genotype on the soil microbiome, metaphenome, and carbon chemistry.

IMPORTANCE: Carbon is cycled through the air, plants, and belowground environment. Understanding soil carbon cycling in deep soil profiles will be important to mitigate climate change. Soil carbon cycling is impacted by water, plants, and soil microorganisms, in addition to soil mineralogy. Measuring biotic and abiotic soil properties provides a perspective of how soil microorganisms interact with the surrounding chemical environment. This study emphasizes the importance of considering biotic interactions with inorganic and oxidizable soil carbon in addition to total organic carbon in carbonate-containing soils for better informing soil carbon management decisions.

Arbuscular mycorrhizae influence raspberry growth and soil fertility under conventional and organic fertilization.

Introduction: Introducing beneficial soil biota such as arbuscular mycorrhizal fungi (AMF) to agricultural systems may improve plant performance and soil fertility. However, whether bioinocula species composition affects plant growth and soil fertility, and whether fertilizer source influences AMF colonization have not been well characterized. The objectives of this research were to: (1) assess if AMF bioinocula of different species compositions improve raspberry (Rubus idaeus L.) performance and characteristics of soil fertility and (2) evaluate the impact of fertilizer source on AMF colonization. Methods: Five bioinocula with different AMF species compositions and three fertilizer sources were applied to tissue culture raspberry transplants in a randomized complete block design with eight replicates. Plants were grown in a greenhouse for 14 weeks and plant growth, tissue nutrient concentrations, soil fertility, and AMF root colonization were measured. Results: Shoot K and Zn concentrations as well as soil pH and K concentration increased in the Commercial Mix 1 treatment (Glomus, Gigaspora, and Paraglomus AMF species) compared to the non-inoculated control. RFI (raspberry field bioinoculum; uncharacterized AMF and other microbiota) increased soil organic matter (SOM), estimated nitrogen release (ENR), and soil copper (Cu) concentration compared to the non-inoculated control. Furthermore, plants receiving the Mix 1 or RFI treatments, which include more AMF species, had greater AMF root colonization than the remaining treatments. Plants receiving organic fertilizer had significantly greater AMF colonization than conventionally fertilized plants. Conclusion: Taken together, our data indicate that coupling organic fertilizers and bioinocula that include diverse AMF species may enhance raspberry growth and soil fertility.

Late-season corn stalk nitrate measurements across the US Midwest from 2006 to 2018.

The late-season Corn Stalk Nitrate Test (CSNT) is a well-known tool to help evaluate the after-the-fact performance of nitrogen management. The CSNT has the unique ability to distinguish between optimal and excessive corn nitrogen status, which makes it helpful for identifying the over-application of N so that farmers can adjust their future nitrogen decisions. This paper presents a multi-year and multi-location dataset of late-season corn stalk nitrate test measurements across the US Midwest from 2006 to 2018. The dataset consists of 32,025 corn stalk nitrate measurements from 10,675 corn fields. The nitrogen form, total N rate applied, US state, year of harvest, and climatic conditions are included for each corn field. When available, previous crop, manure source, tillage, and timing of N application are also informed. We provide a detailed description of the dataset to make it usable by the scientific community. Data are published through an R package and also available at the USDA National Agricultural Library Ag Data Commons repository and through an interactive website.

Screening of Diabetic Nephropathy Progression-Related Genes Based on Weighted Gene Co-expression Network Analysis.

The purpose of this study is to explore the progression-related genes of diabetic nephropathy (DN) through weighted gene co-expression network analysis (WGCNA). The gene expression dataset GSE14202 was downloaded from the GEO database for differential expression analysis. WGCNA v1.69 was used to perform co-expression analysis on differentially expressed genes. 25 modular genes were selected through WGCNA. The motif enrichment analysis was performed on 25 genes, and 34 motifs were obtained, of which 8 transcription factors (TFs) were differentially expressed. GENIE3 was applied to analyze the expression correlation of 8 differentially expressed TFs and 25 genes. Combined with the predicted TF-target gene relationship, 69 interactions between 8 TFs and 18 genes were obtained. The functional enrichment analysis of 18 genes showed that 7 key genes were obviously enriched in adaptive immune response and were clearly up-regulated in advanced DN patients. The expression of C1S, LAIR1, CD84, SIT1, SASH3, and CD180 in glomerular samples from DN patients was significantly up-regulated in compared with normal samples, and the expression of these genes was negatively correlated with GFR. We observed that in the in vitro cell model of DN, the relative expression levels of 5 key genes (except SASH3) were obviously elevated in the high-glucose group. Five key genes were identified to be related to the progression of DN. The findings of this study may provide new ideas and therapeutic targets for exploring the pathogenesis of DN.

Evaluation of real-time nutrient analysis of fertilized raspberry using petiole sap.

The time delay in receiving conventional tissue nutrient analysis results caused red raspberry (Rubus idaeus L.) growers to be interested in rapid sap tests to provide real-time results to guide immediate nutrient management practices. However, sap analysis has never been conducted in raspberry. The present work aimed to evaluate the relationship of petiole sap nitrate (NO3 -), potassium (K+), and calcium (Ca2+) concentrations measured using compact ion meters and leaf tissue total nitrogen (TN), potassium (K), and calcium (Ca) concentrations measured using conventional tissue nutrient analysis. The relationship of petiole sap NO3 - and leaf tissue TN concentrations with plant growth and production variables was also explored. Fertilizer treatments of urea were surface applied to raised beds of established "Meeker" floricane red raspberry plots at control, low, medium, and high rates (0, 34, 67, and 101 kg N ha-1, respectively) in 2019 and 2020. The experiment was arranged in a randomized complete block design with three replications. Whole leaves were collected from representative primocanes in mid- and late- July and August 2019 and 2020 (i.e., four sampling time points per year). At each sampling time point, a subsample of leaves was used for petiole sap analyses of NO3 -, K+, and Ca2+ concentrations using compact ion meters, and conventional tissue testing of leaf tissue TN, K, and Ca concentrations, respectively. There were no interactions between N fertilizer rate and year nor between N fertilizer rate and sampling time. No significant differences were found due to N fertilizer rate for petiole sap NO3 -, K+, Ca2+ nor leaf tissue TN, K, Ca concentrations. However, significant year and sampling time effects occurred in measured petiole sap and leaf tissue nutrient concentrations. Overall, the correlations between petiole sap NO3 - and leaf tissue TN, petiole sap Ca2+ and leaf tissue Ca, petiole sap K+ and leaf tissue K concentrations were non-strong and inconsistent. Future research is warranted as the interpretation of correlations between raspberry petiole sap and leaf tissue nutrient concentrations were inconclusive.

TLR4 activation inhibits the proliferation and osteogenic differentiation of skeletal muscle stem cells by downregulating LGI1

Skeletal muscle stem cells (SMSCs) are vital to the growth, maintenance, and repair of the muscles; emerging evidence has indicated that Toll-like receptor 4 (TLR4) can potentially regulate muscle regeneration. In present study, in vitro and in vivo experiments were performed to explore the correlation of TLR4 with leucine-rich glioma-inactivated 1 (LGI1) as well as their effects on the proliferation and osteogenesis potential of SMSCs. In order to examine the regulatory mechanisms of TLR4 and LGI1 in SMSCs, the obtained cells were treated with lipopolysaccharide (LPS, used as an activator of TLR4) of different concentration at different time points as well as the siRNA against LGI1. Subsequently, a series of detection was undertaken in order to measure the proliferation and differentiation potential of SMSCs, which involved detection of the related factors, cell activity, and the sphere-forming capability. Following LPS treatment, the increased TLR4 expression and reduced LGI1 expression were observed. Consequently, we also discovered that Erk signaling pathway was inactivated and cell proliferation and osteogenesis capabilities declined, presented by the downregulation of related factors such as cyclin B1 and runt-related transcription factor 2. Moreover, the cell activity and sphere-formation performance of SMSCs were also declined. These results were also validated in rats with cecal ligation and perforation-induced rat models with sepsis. In conclusion, the present study reveals a regulatory mechanism in SMSCs whereby LGI1 expression is reduced by TLR4, thus impeding cell proliferation and osteogenesis, highlighting TLR4 as a potential therapeutic target against many diseases related to SMSCs. (AU)

TLR4 activation inhibits the proliferation and osteogenic differentiation of skeletal muscle stem cells by downregulating LGI1.

Skeletal muscle stem cells (SMSCs) are vital to the growth, maintenance, and repair of the muscles; emerging evidence has indicated that Toll-like receptor 4 (TLR4) can potentially regulate muscle regeneration. In present study, in vitro and in vivo experiments were performed to explore the correlation of TLR4 with leucine-rich glioma-inactivated 1 (LGI1) as well as their effects on the proliferation and osteogenesis potential of SMSCs. In order to examine the regulatory mechanisms of TLR4 and LGI1 in SMSCs, the obtained cells were treated with lipopolysaccharide (LPS, used as an activator of TLR4) of different concentration at different time points as well as the siRNA against LGI1. Subsequently, a series of detection was undertaken in order to measure the proliferation and differentiation potential of SMSCs, which involved detection of the related factors, cell activity, and the sphere-forming capability. Following LPS treatment, the increased TLR4 expression and reduced LGI1 expression were observed. Consequently, we also discovered that Erk signaling pathway was inactivated and cell proliferation and osteogenesis capabilities declined, presented by the downregulation of related factors such as cyclin B1 and runt-related transcription factor 2. Moreover, the cell activity and sphere-formation performance of SMSCs were also declined. These results were also validated in rats with cecal ligation and perforation-induced rat models with sepsis. In conclusion, the present study reveals a regulatory mechanism in SMSCs whereby LGI1 expression is reduced by TLR4, thus impeding cell proliferation and osteogenesis, highlighting TLR4 as a potential therapeutic target against many diseases related to SMSCs.

The α2-Plasmin Inhibitor-Plasmin Complex is a Potential Biomarker of Venous Thromboembolism in Orthopedic Trauma Patients.

BACKGROUND: Venous thromboembolism (VTE) is a common complication in orthopedic trauma patients. Accurate prediction of individual thrombosis risk is important in determining whether prophylactic treatment with anticoagulants is necessary. In this study, we screened for biomarkers that could be used as predictors of VTE risk and evaluated their efficacy and benefit in treating orthopedic traumatic patients. METHODS: A total of 683 patients with orthopedic trauma were consecutively enrolled between January 2017 and June 2018 at Renmin Hospital of Wuhan University. Demographic and clinical information was collected, and VTE risk was assessed using the Caprini risk assessment score. The concentrations of PIC, coagulation parameters and other routine biochemical markers were quantified. The Mann-Whitney U test was used to identify potential biomarkers which were significantly different between patients who developed VTE and those who did not. Correlation between individual parameters was assessed using Pearson's correlation. A nomogram model was constructed to predict VTE risk using a combination of biomarkers, and a decision curve analysis was performed to assess the net benefit of using each biomarker. RESULTS: Patients with VTE had significantly higher levels of PIC (p = 0.037) and DD (p = 0.042) than those without, even after adjusting for confounding factors. PIC and DD levels increased in a stepwise fashion with increasing VTE risk and were the markers most strongly associated with Caprini score (PIC, r = 0.408; DD, r = 0.474; p < 0.001). In decision curve analysis, PIC levels provided greater net benefit than the Caprini score or DD level across patients with various bleeding risks. CONCLUSIONS: Plasma PIC levels are a useful biomarker of VTE risk and can be used to determine whether pharmaco-prophylaxis is needed in orthopedic trauma patients.

Expression of miR-409-5p in gestational diabetes mellitus and its relationship with insulin resistance.

Expression of miR-409-5p in gestational diabetes mellitus (GDM) and its relationship with insulin resistance were explore. One hundred and forty-nine pregnant women who underwent antenatal examination in Taizhou First People's Hospital were divided into a GDM group and a control group according to whether they had GDM or not. Serum miR-409-5p expression of the two groups was detected, and the levels of glycosylated hemoglobin (HbAlc) and other GDM-related biochemical indicators were measured. Fasting plasma glucose (FPG) was determined by glucose oxidase method, fasting insulin (FINS) was detected by radioimmunoassay, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. The relationship between miR-409-5p and other biochemical indicators and insulin resistance was analyzed, and logistic multivariate regression was employed to analyze the risk factors of GDM. miR-409-5p was highly expressed in the serum of GDM patients. HbAlc, FPG, FINS, and HOMA-IR in pregnant women in the GDM group were markedly higher than those in the control group. The serum miR-409-5p in GDM pregnant women showed a positive correlation with HbAlc, FPG, FINS, and HOMA-IR (P<0.05). The insulin resistance group presented remarkably higher serum miR-409-5p level than the non-insulin resistance group. Moreover, it was found that elevated miR-409-5p, FINS, and HOMA-IR were all independent risk factors for the onset of GDM. miR-409-5p is highly expressed in the serum of patients with GDM, and it is positively correlated with insulin resistance index of GDM patients, which may be a potential target for clinical diagnosis and treatment of GDM.

A-kinase interacting protein 1 as a potential biomarker of advanced tumor features and increased recurrence risk in papillary thyroid carcinoma patients.

BACKGROUND: This study aimed to detect the expression of A-kinase interacting protein 1 (AKIP1) and explore its correlation with clinicopathological features and clinical outcomes in papillary thyroid carcinoma (PTC) patients. METHODS: A total of 245 PTC patients treated by lobectomy or thyroidectomy were analyzed in this retrospective study. AKIP1 expression in tumor and adjacent tissue (from Specimen Room of our hospital) was detected by immunohistochemical (IHC) assay and then categorized as four grades: AKIP1 low (IHC score ≤3), high+ (IHC score 4-6), high++ (IHC score 7-9), and high+++ (IHC score 10-12). RESULTS: A-kinase interacting protein 1 low, high+, high++, and high+++ expression was 101 (41.2%), 101 (41.2%), 32 (13.1%), and 11 (4.5%) in tumor tissues, while was 173 (70.6%), 61 (24.9%), 9 (3.7%), and 2 (0.8%) in adjacent tissues. Further comparison analysis showed increased grade of AKIP1 expression in tumor tissues compared to adjacent tissue. Meanwhile, increased grade of tumor AKIP1 expression was correlated with larger tumor size, extrathyroidal invasion, increased pT stage, and higher pTNM stage. For prognosis, increased grade of tumor AKIP1 expression was correlated with shorter disease-free survival (DFS), while was not correlated with overall survival (OS). Forward stepwise multivariate Cox's regression revealed that higher tumor AKIP1 was an independent factor predicting worse DFS, but not OS. CONCLUSION: AKIP1 is upregulated in tumor tissue, and increased tumor AKIP1 expression correlates with advanced tumor features and increased recurrence risk in PTC patients, which suggest that AKIP1 severs as a potential marker for effective supervision of PTC progression.

Comparison of subtotal vertebral resection with reconstruction and percutaneous vertebroplasty for treatment of metastasis in the lumbar spine.

Purpose: Tumor metastasis in the spine can cause pain and fractures, leading to deformities, and deficits in movement, sensation, and bowel/bladder function. Percutaneous vertebroplasty (PVP) and subtotal vertebral resection with reconstruction (SVR) are suitable treatments, but their relative clinical efficacy is uncertain. The purpose of this retrospective cohort study was to compare the management and clinical effect of SVR for lumbar metastatic tumor with PVP.Methods: Sixty-seven patients (mean age: 58.6 years) with metastases in the lumbar spine received SVR or PVP at our institution between 2010 and 2013. Thirty-three patients received SVR via a posterior approach, in which vertebrae were resected, with the anterior and lateral walls retained using polymethylmethacrylate (PMMA), followed by reconstruction and pedicle screw fixation. Thirty-four patients received PVP via the vertebral pedicle. Patients were followed for 3-26 months.Results: None of the patients experienced serious complications after surgery, and all patients experienced significant amelioration of pain. Twelve patients (8 in the PVP group and 4 in the SVR group) died during the follow-up, and the survival time was significantly longer in the SVR group. Two patients in the SVR group and 7 patients in the PVP groups experienced recurrence during follow-up, but the groups had no significant difference in local recurrence. Both treatments significantly reduced scores for pain on a visual analog scale (pain-VAS) and disability (Oswestry Disability Index [ODI]), and increased performance status (Karnofsky Performance Status [KPS]). Compared with the PVP group, the SVR group had better ODI score at 1 month and 3 months after surgery and a higher KPS score at 1 month after surgery. The two groups had no significant difference in pain-VAS scores during follow-up.Conclusions: SVR is a reliable treatment for lumbar metastatic tumor and provides good survival rate and satisfying follow-up results.

Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting RORß.

BACKGROUND: Colorectal cancer remains one of the most common malignant tumors worldwide. Colorectal cancer initiating cells (CCICs) are a small subpopulation responsible for malignant behaviors of colorectal cancer. Aberrant activation of the Wnt pathways regulates the self-renewal of CCIC. However, the underlying mechanism(s) remain poorly understood. METHODS: Via retroviral library screening, we identified Nuclear Receptor-Interacting Protein 2 (NRIP2) as a novel interactor of the Wnt pathway from enriched colorectal cancer colosphere cells. The expression levels of NRIP2 and retinoic acid-related orphan receptor ß (RORß) were further examined by FISH, qRT-PCR, IHC and Western blot. NRIP2 overexpressed and knockdown colorectal cancer cells were produced to study the role of NRIP2 in Wnt pathway. We also verified the binding between NRIP2 and RORß and investigated the effect of RORß on CCICs both in vitro and in vivo. Genechip-scanning speculated downstream target HBP1. Western blot, ChIP and luciferase reporter were carried to investigate the interaction between NRIP2, RORß, and HBP1. RESULTS: NRIP2 was significantly up-regulated in CCICs from both cell lines and primary colorectal cancer tissues. Reinforced expression of NRIP2 increased Wnt activity, while silencing of NRIP2 attenuated Wnt activity. The transcription factor RORß was a key target through which NRIP2 regulated Wnt pathway activity. RORß was a transcriptional enhancer of inhibitor HBP1 of the Wnt pathway. NRIP2 prevented RORß to bind with downstream HBP1 promoter regions and reduced the transcription of HBP1. This, in turn, attenuated the HBP1-dependent inhibition of TCF4-mediated transcription. CONCLUSIONS: NRIP2 is a novel interactor of the Wnt pathway in colorectal cancer initiating cells. interactions between NRIP2, RORß, and HBP1 mediate a new mechanism for CCIC self-renewal via the Wnt activity.

[Pyrroloquinoline quinone inhibited oxidative stress induced-apoptosis of Schwann cells via mitochondrial apoptotic signaling pathway in vitro].

Objective: To investigate the mechanisms of Pyrroloquinoline quinone (PQQ) against oxidative stress induced apoptosis in Schwann cells (SCs). Methods: SCs were cultured in vitro, identified by S-100 immunofluorence staining. SCs were divided into control group,H2O2 induced group,H2O2 + PQQ treated group.CCK-8 assay was used to detect cell proliferation. Apoptosis was detected by flow cytometry with Annecin V-FITC/PI staining, mitochondrial transmembrane potential was detected by flow cytometry with JC-1 labeled staining, cytochrome C (CytC), Bax and Caspase-9 protein levels was detected by Western blot analysis. Results: In this study, the S-100 positive cells were more than 95％,cell proliferation was decreased in H2O2 induced SCs,apoptotic rate was increased, mitochondrial transmnembrane potential was decreased,CytC, Bax and Caspase-9 protein levels were increased. After PQQ added, cell proliferation was increased, apoptotic rate decreased, mitochondrial transmembrane potential increased, CytC, Bax and Caspase-9 protein levels decreased. Conclusions: PQQ protects SCs from oxidative induced apoptosis by inhibiting mitochondrial signaling pathway.

Retrospective analysis of surgical treatment and postoperative follow-up study of adult primary intraspinal tumors / 中国肿瘤临床

Objective:To evaluate the diagnosis, surgical treatment, and neurological function recovery after surgery of patients with intraspinal tumors. Methods:The clinical data of 69 patients who suffered from intraspinal tumors and underwent surgery from Janu-ary 2008 to December 2012 were retrospectively analyzed. Neuroimaging and ASIA scoring were performed to examine the pathologi-cal characteristics of tumors and the neurological function of these patients before and after treatment. The major factors affecting prognosis were also probed, and the average follow-up period was 12.2 months. Results:Of the total cases, 62.3%showed intradural extramedullary intraspinal tumors located in the thoracic vertebra. Neurilemoma (Schwannoma) and meningioma were the most com-mon pathological types (53.5%). Posterior approaches with hemi-and complete-laminectomy were conducted to expose the intraspi-nal tumors, and the separation and removal of the tumors located at the cervical and thoracic levels were aided with surgical microsco-py. The main clinical symptoms, including back pain, radicular neuralgia, sensory disturbance, and motor dysfunction, were significant-ly improved after surgery, and this observation was supported by the follow-up ASIA scores before and after treatment. Of the in-volved cases, 91%were successfully treated, and their tumors were totally resected. Conclusion:Total or subtotal intraspinal tumor re-section enhanced with surgical microscopy could achieve satisfactory clinical results through posterior hemi-or complete-laminectomy.

Protection of carboxymethylated chitosan on chondrocytes from nitric oxide-induced apoptosis by regulating phosphatidylinositol 3-kinase/Akt signaling pathway.

Chondrocyte apoptosis is the most important element of development and progression of osteoarthritis (OA). Nitric oxide (NO) was used as the agent to induce chondrocyte apoptosis. Carboxymethylated chitosan (CMCS) has anti-apoptosis effect on many cell types in vitro. This study was designed to investigate the protective effect of CMCS on NO-induced chondrocyte apoptosis and the probable molecular mechanisms. The newborn Sprague-Dawley (SD) rats were used in this study for isolation of chondrocytes. The cell viability was determined by cell counting kit (CCK-8), cell apoptosis was detected by Annexin-V/PI double staining assay kit. The levels of phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt), Bcl-2 and Bax were determined by Western blot analysis. The caspase-3 activity was determined by a quantitative colorimetric assay. Results showed that pretreatment with CMCS could inhibit the apoptosis induced by NO. CMCS could decrease the activity of NO and decrease the expression of Bcl-2, p-PI3K and p-Akt, increase the expression of Bax, cytochrome c and caspase-3. CMCS also could reverse the effect of NO that prompted matrix metalloproteinase-13 (MMP-13) and inhibited tissue inhibitor of metalloproteinase-1 (TIMP-1) activity. All the present results indicated that CMCS can protect NO induced chondrocytes apoptosis by activate PI3K/Akt signaling pathway.

Berberine promotes proliferation of sodium nitroprusside-stimulated rat chondrocytes and osteoarthritic rat cartilage via Wnt/ß-catenin pathway.

Berberine chloride (BBR) is an isoquinoline derivative alkaloid isolated from medicinal herbs, including Coptis chinensis and Berberis aristate. This compound plays significant roles in the treatment of osteoarthritis (OA). The purpose of this study was to investigate the effects of BBR on the proliferation of sodium nitroprusside (SNP)-stimulated chondrocytes in vitro, the articular cartilage in a rat OA model, as well as to discuss the molecular mechanisms underlying these effects. In vitro, we demonstrated that BBR led to cell proliferation, increased the cell population in S-phase and decreased that in G0/G1-phase; moreover, the F-actin remodeling in SNP-stimulated chondrocytes were prevented. In addition, BBR markedly up-regulated ß-catenin, c-Myc, and cyclin D1 expression of genes and proteins, and down-regulated glycogen synthase kinase-3ß (GSK-3ß) and matrix metalloproteinase-7 (MMP-7) expression. Notably, inhibition of the Wnt/ß-catenin pathway by XAV939 partially blocked these effects. The in vivo results suggested that BBR promoted ß-catenin protein level and enhanced proliferating cell nuclear antigen (PCNA) expression in osteoarthritic rat cartilage. In conclusion, these findings indicate that BBR promotes SNP-stimulated chondrocyte proliferation by promoting G1/S phase transition and synthesis of PCNA in cartilage through activation of Wnt/ß-catenin signaling pathway.

Carboxymethylated chitosan protects rat chondrocytes from NO­induced apoptosis via inhibition of the p38/MAPK signaling pathway.

In the present study, the effect of carboxymethylated chitosan (CMCS) on nitric oxide (NO)­induced apoptosis, and activation of the p38/MAPK signaling pathway in chondrocytes were investigated. Cartilage was isolated from the knee joints of Sprague­Dawley rats, and was used to establish cultured primary chondrocytes. The chondrocytes were incubated with the NO donor, sodium nitroprusside (SNP), to induce apoptosis, and were treated with CMCS and the p38 inhibitor, SB203580. Cell viability was assessed using a Cell Counting Kit­8 assay. Apoptosis of the chondrocytes was detected using Annexin V­fluorescein isothiocyanate/propidium iodide staining. The activation of p38 was detected using Western blot analysis, and caspase­3 activity was detected using a caspase­3 detection kit. The results indicated that, in chondrocytes treated with SNP, the optical density values of the experimental groups were significantly lower, compared with the control group (P<0.05). The exposure of the cells to CMCS significantly prevented apoptosis (P<0.05), and a dose­dependent effect was demonstrated using fluorescence­activated cell sorting analysis (P<0.05). Examination of the expression and activity of p38 and caspase­3, respectively, showed that SNP increased the expression of p38 and activity of caspase­3, and this trend was reversed following the addition of CMCS and SB203580. Taken together, these findings indicated that CMCS prevented NO­induced apoptosis of chondrocytes via inhibition of the p38/mitogen­activated protein kinase signaling pathway in vitro.

Protective effect of carboxymethylated chitosan on hydrogen peroxide-induced apoptosis in nucleus pulposus cells.

Although the etiology of intervertebral disc degeneration is poorly understood, one approach to prevent this process may be to inhibit apoptosis. In the current study, the anti­apoptotic effects of carboxymethylated chitosan (CMCS) in nucleus pulposus (NP) cells were investigated with the aim to enhance disc cell survival. Rat NP cells were isolated and cultured in vitro, and hydrogen peroxide (H2O2) was used to build the NP cell apoptosis model. Cell viability was assessed with a cell counting kit­8 assay. The ratio of apoptotic cells was surveyed by annexin V­fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining analysis, and the morphology was observed by Hoechst 33342 staining. The mitochondrial membrane potential of NP cells was evaluated by rhodamine 123 fluorescence staining. Reverse transcription (RT)­quantitative polymerase chain reaction (qPCR) was performed to measure mRNA levels of inducible nitric oxide synthase (iNOS), caspase­3, B­cell lymphoma (Bcl)­2, type II collagen and aggrecan. Western blot analysis was performed to detect protein levels of iNOS and Bcl­2. The annexin V­FITC/PI and Hoechst 33342 staining results indicated that CMCS was able to prevent NP cells from apoptosis in a dose­dependent manner. Rhodamine 123 staining clarified that CMCS reduced the impairment of the mitochondrial membrane potential in H2O2­treated NP cells. Reduced caspase­3 and increased Bcl­2 activity were detected in CMCS­treated NP cells by RT­qPCR and western blot analysis. CMCS also promoted the proliferation and secretion of type II collagen and aggrecan in H2O2­treated NP cells. CMCS was indicated to be effective in preventing apoptotic cell death in vitro, demonstrating the potential advantages of this therapeutic approach in regulating disc degeneration.

Neuroprotective effects of carboxymethylated chitosan on hydrogen peroxide induced apoptosis in Schwann cells.

The protective and promotion effects of Carboxymethylated chitosan (CMCS) on peripheral nerve and cultured Schwann cells (SCs) have been demonstrated, but few studies discussed the protective roles of CMCS on SCs apoptosis. We explored the anti-apoptotic activities of CMCS in SCs to enhance cells survival in this present study. Rat SCs were isolated and cultured in vitro, hydrogen peroxide (H2O2) was used to establish the apoptosis models of SCs. Cells proliferative activity was assessed by CCK-8 assay. The apoptosis of SCs was detected by flow cytometry (FCM) analysis. Superoxide dismutase (SOD) and malondialdehyde (MDA) activities were detected by the corresponding assay kit. The nuclear appearance of apoptotic SCs was observed by nuclear staining with Hoechst 33342. The real-time PCR was performed to detect the levels of Bcl-2, Bax, Caspase-3 and -9 mRNA. Detection of caspase-3 and -9 was fulfilled by using Western blot analysis. FCM assay and Hoechst33342 staining results indicated that CMCS could protect SCs from apoptosis with dose and time-dependent manner. SOD and MDA analysis results indicated that CMCS could promote SOD activity and reduce the MDA levels in H2O2 induced SCs. The decreased caspase-3, -9 and Bax activities and increased Bcl-2 activity were observed in CMCS treated SCs. The present study indicates CMCS has the neuroprotective effect on peripheral nerves and inhibit SCs apoptosis.

[Effects of pyrroloquinoline quinine on oxidative stress-induced apoptosis of Schwann cells and its mechanism].

OBJECTIVE: To investigate the effects of Pyrroloquinoline quinine (PQQ) on hydrogen peroxide-induced apoptosis of Schwann cells (SCs) and its mechanism. METHODS: SCs were isolated and cultured in vitro, and identified by S-100 immunofluorescence staining. The cultured SCs were divided into control group, hydrogen peroxide-treated group, hydrogen peroxide and PQQ treated groups. The intracellular superoxide dismutase (SOD) and malondialdehyde (MDA) content was detected; the apoptotic rate of SCs induced by hydrogen peroxide was determined by flow cytometry assay. The Hoechst33342 staining was used to detect the nuclear fragmentation and apoptotic nuclear condensation of SCs; the Rhodamine123 staining was used to detect the changes of mitochondrial membrane potential in SCs, the Western blot analysis was used to detect the expression of Bcl-2 in hydrogen peroxide induced SCs. RESULTS: The SOD activity was significantly decreased and MDA level was increased in H2O2 induced SCs (P < 0.05), after addition of PQQ, the SOD content increased and MDA content decreased (P < 0.05). Flow cytometry results showed that the early apoptotic rate was 58.8% in H2O2 induced SCs, which has significant difference compared with the control group (P < 0.05), after addition of 10, 50, 100 nmol/L PQQ, the apoptotic rates were reduced to 33.7%, 18.7%, 3.9% respectively, showing significantly different with injured group (P < 0.05). Hoechst 33342 staining showed that H2O2 induced SCs had typical morphological characteristics, such as uptake of nuclear chromatin, nuclear shrinkage, nuclear fragmentation phenomenon. The proportion of apoptotic cells after PQQ treatment reduced. Rhodamine staining results showed that the H2O2 induced mitochondrial membrane potential reduction in SCs, which was reversed by addition of PQQ. Western blot analysis showed that the expression of Bcl-2 was decreased in H2O2 induced SCs, while it increased significantly after addition of PQQ (P < 0.05). CONCLUSION: PQQ has a protective effect on oxidative stress-induced apoptosis of SCs.