Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer.

BACKGROUND & AIMS: Both nonalcoholic fatty liver disease (NAFLD) and colorectal cancer (CRC) are prevalent worldwide. The effects of concomitant NAFLD on the risk of colorectal liver metastasis (CRLM) and its mechanisms have not been definitively elucidated. METHODS: We observed the effect of concomitant NAFLD on CRLM in the mouse model and explored the underlying mechanisms of specific myeloid-derived suppressor cells (MDSCs) recruitment and then tested the therapeutic application based on the mechanisms. Finally we validated our findings in the clinical samples. RESULTS: Here we prove that in different mouse models, NAFLD induces F4/80+ Kupffer cells to secret chemokine CXCL5 and then recruits CXCR2+ MDSCs to promote the growth of CRLM. CRLM with NAFLD background is refractory to the anti-PD-1 monoclonal antibody treatment, but when combined with Reparixin, an inhibitor of CXCR1/2, dual therapy cures the established CRLM in mice with NAFLD. Our clinical studies also indicate that fatty liver diseases increase the infiltration of CXCR2+ MDSCs, as well as the hazard of liver metastases in CRC patients. CONCLUSIONS: Collectively, our findings highlight the significance of selective CXCR2+/CD11b+/Gr-1+ subset myeloid cells in favoring the development of CRLM with NAFLD background and identify a pharmaceutical medicine that is already available for the clinical trials and potential treatment.

Neoadjuvant tislelizumab plus stereotactic body radiotherapy and adjuvant tislelizumab in early-stage resectable hepatocellular carcinoma: the Notable-HCC phase 1b trial.

Notable-HCC (NCT05185531) is a phase 1b trial, aiming to evaluate the safety and preliminary effectiveness of neoadjuvant PD-1 blockade plus stereotactic body radiotherapy (SBRT) in early-stage resectable hepatocellular carcinoma (HCC). Twenty patients with HCC of BCLC stage 0-A received 3 × Gy SBRT and two cycles of tislelizumab, an anti-PD-1 monoclonal antibody before the curative HCC resection. Primary endpoints were the surgery delay, radiographic and pathological tumor response after the neoadjuvant therapy, safety and tolerability. During the neoadjuvant therapy, treatment-related adverse events (TRAEs) of grade 1-2 occurred in all 20 patients (100%), eight patients (40%) had grade 3 TRAEs, no grade 4 to 5 TRAE occurred, and all resolved without corticosteroids treatment. Per mRECIST, the objective response rate was 63.2% (12/19), with 3 complete response; the disease control rate was 100%. Two (10.5%) patients achieved complete pathological response. No surgery delay occurred. The neoadjuvant therapy did not increase the surgical difficulty or the incidence of complications. Secondary endpoints of disease-free survival and overall survival were not mature at the time of the analysis. Our pilot trial shows that neoadjuvant therapy with anti-PD-1 + SBRT is safe and promotes tumor responses in early-stage resectable HCC.

Beyond Small Molecules: Antibodies and Peptides for Fibroblast Activation Protein Targeting Radiopharmaceuticals.

Fibroblast activation protein (FAP) is a serine protease characterized by its high expression in cancer-associated fibroblasts (CAFs) and near absence in adult normal tissues and benign lesions. This unique expression pattern positions FAP as a prospective biomarker for targeted tumor radiodiagnosis and therapy. The advent of FAP-based radiotheranostics is anticipated to revolutionize cancer management. Among various types of FAP ligands, peptides and antibodies have shown advantages over small molecules, exemplifying prolonged tumor retention in human volunteers. Within its scope, this review summarizes the recent research progress of the FAP radiopharmaceuticals based on antibodies and peptides in tumor imaging and therapy. Additionally, it incorporates insights from recent studies, providing valuable perspectives on the clinical utility of FAP-targeted radiopharmaceuticals.

Electroacupuncture Mediates Fat Metabolism and Autophagy via a Sirt3-Dependent Mechanism in Mice Fed High-Fat Diet.

This study investigates the therapeutic potential of electroacupuncture (EA) on obesity, focusing on its influence on autophagy and energy metabolism, utilizing a high-fat diet (HFD)-induced mouse model. Treatment with EA significantly reduces body weight, fat deposition, and lipid accumulation in HFD-fed mice. Additionally, EA effectively ameliorates metabolic imbalances, reducing blood glucose levels and plasma markers of liver function. At the molecular level, EA enhances the expression of thermogenesis-associated genes in brown adipose tissue and decreases p53 expression, suggesting a decrease in apoptosis. Autophagy in white adipose tissue is inhibited by EA, as demonstrated by the suppression of key autophagy-related proteins. Further experiments highlight the critical role of Sirtuin 3 (Sirt3) in EA's anti-obesity effects. Sirt3 supplementation combined with EA results in reduced body weight, fat deposition, and lipid accumulation, along with modulations in key metabolic indicators. Moreover, EA's modulatory effect on uncoupling protein 1 (Ucp1), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α), and p53 is found to be Sirt3 dependent. In conclusion, EA exerts beneficial effects against obesity through Sirt3-dependent modulation of autophagy and energy metabolism, indicating a potential therapeutic approach for obesity and related metabolic disorders.

Marriage of radiotracers and total-body PET/CT rapid imaging system: current status and clinical advances.

Radiotracers and medical imaging equipment are the two main keys to molecular imaging. While radiotracers are of great interest to research and industry, medical imaging equipment technology is blossoming everywhere. Total-body PET/CT (TB-PET/CT) has emerged in response to this trend and is rapidly gaining traction in the fields of clinical oncology, cardiovascular medicine, inflammatory/infectious diseases, and pediatric diseases. In addition, the use of a growing number of radiopharmaceuticals in TB-PET/CT systems has shown promising results. Notably, the distinctive features of TB-PET/CT, such as its ultra-long axial field of view (194 cm), ultra-high sensitivity, and capability for low-dose tracer imaging, have enabled enhanced imaging quality while reducing the radiation dose. The envisioned whole-body dynamic imaging, delayed imaging, personalized disease management, and ultrafast acquisition for motion correction, among others, are achieved. This review highlights two key factors affecting molecular imaging, describing the rapid imaging effects of radiotracers allowed at low doses on TB-PET/CT and the improvements offered compared to conventional PET/CT.

Human umbilical cord mesenchymal stem cells-derived exosomes exert anti-inflammatory effects on osteoarthritis chondrocytes.

Inflammation of chondrocytes plays a critical role in the occurrence and development of osteoarthritis (OA). Recent evidence indicated exosomes derived from mesenchymal stem cells (MSCs-Exos) exhibit excellent anti-inflammatory ability in many troublesome inflammatory diseases including OA. In the present study, we aimed to explore the role of human umbilical cord-derived MSCs-Exos (hUC-MSCs-Exos) in treating the inflammation of chondrocytes and its related mechanisms. Ultracentrifugation was applied to isolate hUC-MSCs-Exos from the culture supernatant of hUC-MSCs. Two OA-like in vitro inflammation models of human articular chondrocytes induced with interleukin 1ß (IL-1ß) and co-incubation with macrophage utilizing transwell cell culture inserts were both used to evaluate the anti-inflammatory effects of hUC-MSCs-Exos. The mRNA sequencing of chondrocytes after treatment and microRNA (miRNA) sequencing of hUC-MSCs-Exos were detected and analyzed for possible mechanism analysis. The results of the study confirmed that hUC-MSCs-Exos had a reversed effect of IL-1ß on chondrocytes in the expression of collagen type II alpha 1 (COL2A1) and matrix metalloproteinase 13 (MMP13). The addition of hUC-MSCs-Exos to M1 macrophages in the upper chamber showed down-regulation of IL-1ß and tumor necrosis factor α (TNF-α), up-regulation of IL-10 and arginase1 (ARG1), and reversed the gene and protein expression of COL2A1 and MMP13 of the chondrocytes seeded in the lower chamber. The results of this study confirmed the anti-inflammatory effects of hUC-MSCs-Exos in the human articular chondrocytes inflammation model. hUC-MSCs-Exos may be used as a potential cell-free treatment strategy for chondrocyte inflammation in OA.

Three-dimensional simulation/printing-assisted surgery for symptomatic metastatic epidural spinal cord compression of posterior column: efficacy assessment based on 2-year follow-up.

Background: Surgical intervention is necessary for resolving the symptoms of the spinal cord and nerve compression caused by symptomatic metastatic epidural spinal cord compression. However, surgeons are constantly seeking ways to improve surgical efficiency and safety. This study aims to evaluate the efficacy of 3D simulation/printing-assisted surgery for symptomatic metastatic epidural spinal cord compression of the posterior column. Methods: We retrospectively analyzed the clinical data of patients who underwent surgical treatment for symptomatic metastatic epidural spinal cord compression of the posterior column in our hospital from January 2015 to January 2020. The simulated group underwent a 3D digital simulation of the lesion area using imaging data before surgery. Twelve patients in the simulated group also received 3D printing, while the direct surgery group did not receive any 3D simulation or printing. All patients were followed up for at least 2 years. We collected clinical data, including operation time, intraoperative blood loss, pedicle screw adjustment rate, intraoperative fluoroscopy times, the incidence of dural injury and cerebrospinal fluid leakage, VAS score, postoperative neurological function improvement, and tumor recurrence. Statistical analysis was performed using SPSS23.0, and P < 0.05 was considered statistically significant. Results: A total of 46 patients were included in this study, with 20 in the simulated group and 26 in the non-simulated group. The simulated group had better operation time, intraoperative blood loss, screw adjustment rate, fluoroscopy times, and incidence of dural injury/cerebrospinal fluid leakage compared to the non-simulated group. The VAS scores of the two groups improved significantly after the operation and at the last follow-up compared to before the operation. However, there was no statistically significant difference between the two groups. There was also no statistically significant difference in neurological function improvement between the two groups. In the simulated group, 25% of patients relapsed, while in the non-simulated group, 34.61% of patients relapsed. However, there was no statistical difference between the two groups. Conclusion: Preoperative 3D simulation/printing-assisted surgery is a practical and feasible approach for treating symptomatic metastatic epidural spinal cord compression of the posterior column.

Real-world practice of conversion surgery for unresectable hepatocellular carcinoma - a single center data of 26 consecutive patients.

AIM: To understand the proportion of uHCC (unresectable hepatocellular carcinoma) patients who achieve successful conversion resection in a high-volume setting with state of the art treatment options. METHODS: We retrospectively reviewed all HCC patients hospitalized to our center from June 1st, 2019 to June 1st, 2022. Conversion rate, clinicopathological features, response to systemic and/or loco-regional therapy and surgical outcomes were analyzed. RESULTS: A total of 1,904 HCC patients were identified, with 1672 patients receiving anti-HCC treatment. 328 patients were considered up-front resectable. Of the remaining 1344 uHCC patients, 311 received loco-regional treatment, 224 received systemic treatment, and the remainder (809) received combination systemic plus loco-regional treatment. Following treatment, one patient from the systemic group and 25 patients from the combination group were considered to have resectable disease. A high objective response rate (ORR) was observed in these converted patients (42.3% under RECIST v1.1 and 76.9% under mRECIST criteria). The disease control rate (DCR) reached 100%. 23 patients underwent curative hepatectomy. Major post-operative morbidity was equivalent in the both groups (P=0.76). Pathologic complete response (pCR) was 39.1%. During conversion treatment, grade 3 or higher treatment-related adverse events (TRAEs) were observed in 50% of patients. The median follow-up time was 12.9 months (range, 3.9~40.6) from index diagnosis and 11.4 months (range, 0.9~26.9) from resection. Three patients experienced disease recurrence following conversion surgery. CONCLUSIONS: By intensive treatment, a small sub-group of uHCC patients (2%) may potentially be converted to curative resection. Loco-regional combined with systemic modality was relative safe and effective in the conversion therapy. Short-term outcomes are encouraging, but long-term follow-up in a larger patient population are required to fully understand the utility of this approach.

Protective effects of polydatin against bone and joint disorders: the in vitro and in vivo evidence so far.

Polydatin is an active polyphenol displaying multifaceted benefits. Recently, growing studies have noticed its potential therapeutic effects on bone and joint disorders (BJDs). Therefore, this article reviews recent in vivo and in vitro progress on the protective role of polydatin against BJDs. An insight into the underlying mechanisms is also presented. It was found that polydatin could promote osteogenesis in vitro, and symptom improvements have been disclosed with animal models of osteoporosis, osteosarcoma, osteoarthritis and rheumatic arthritis. These beneficial effects obtained in laboratory could be mainly attributed to the bone metabolism-regulating, anti-inflammatory, antioxidative, apoptosis-regulating and autophagy-regulating functions of polydatin. However, studies on human subjects with BJDs that can lead to early identification of the clinical efficacy and adverse effects of polydatin have not been reported yet. Accordingly, this review serves as a starting point for pursuing clinical trials. Additionally, future emphasis should also be devoted to the low bioavailability and prompt metabolism nature of polydatin. In summary, well-designed clinical trials of polydatin in patients with BJD are in demand, and its pharmacokinetic nature must be taken into account.

In-Situ Oxidative Polymerization of Pyrrole Composited with Cellulose Nanocrystal by Reactive Ink-Jet Printing on Fiber Substrates.

A simple and novel method for the deposition of polypyrrole (PPy) and cellulose nanocrystal (CNC) composites on different fiber substrates by reactive ink-jet printing was proposed. PPy/CNCs composites were successfully prepared, and the surface resistance of conductive layer deposited on different fiber substrates is the least when the monomer concentration is 0.6 M. PPy/CNCs were deposited on polyethylene terephthalate (PET) to form a conductive layer by adding polyvinyl alcohol (PVA), and the optimum sintering temperature is 100 °C (monomer/PVA ratio 4.0, conductivity 0.769 S cm-1). The PPy/CNCs conductive layer deposited on the paper has the lowest surface resistance and the best adhesion, and the surface resistance of PPy/CNCs conductive layer decreases first and then increases with the increase of sulfonate concentration. Moreover, the volume of anion in sulfonate will affect the arrangement and aggregation of PPy molecular chain in composite materials. Appropriate sulfonate doping can improve the conductivity and stability of conductive paper, and the maximum conductivity is 0.813 S cm-1. Three devices based on PPy/CNCs conductive paper were proposed and fabricated. Therefore, this ink-jet printing provides a new method for the preparation of conductive materials, sensors, energy storage and electromagnetic shielding, etc.

Extracellular vesicles from human umbilical cord mesenchymal stem cells reduce lipopolysaccharide-induced spinal cord injury neuronal apoptosis by mediating miR-29b-3p/PTEN.

OBJECTIVE: This study investigated the molecular mechanism of whether hUC-MSCs-EVs repressed PTEN expression and activated the PI3K/AKT pathway through miR-29b-3p, thus inhibiting LPS-induced neuronal injury. METHODS: hUC-MSCs were cultured and then identified. Cell morphology was observed. Alizarin red, oil red O, and alcian blue staining were used for inducing osteogenesis, adipogenesis, and chondrogenesis. EVs were extracted from hUC-MSCs and identified by transmission electron microscope observation and Western blot. SCI neuron model was established by 24h lipopolysaccharide (LPS) induction. After the cells were cultured with EVs without any treatment, uptake of EVs by SCI neurons, miR-29b-3p expression, cell viability, apoptosis, caspase-3, cleaved caspase-3, caspase 9, Bcl-2, PTEN, PI3K, AKT, and p-Akt protein levels, caspase 3 and caspase 9 activities, and inflammatory factors IL-6 and IL-1ß levels were detected by immunofluorescence labeling, RT-qPCR, MTT, flow cytometry, Western blot, caspase 3 and caspase 9 activity detection kits, and ELISA. The binding sites between PTEN and miR-29b-3p were predicted by the database and verified by dual-luciferase assay. RESULTS: LPS-induced SCI cell model was successfully established, and hUC-MSCs-EVs inhibited LPS-induced apoptosis of injured spinal cord neurons. EVs transferred miR-29b-3p into LPS-induced injured neurons. miR-29b-3p silencing reversed EV effects on reducing LPS-induced neuronal apoptosis. miR-29b-3p reduced LPS-induced neuronal apoptosis by targeting PTEN. After EVs-miR-inhi and si-PTEN treatment, inhibition of the PI3K/AKT pathway reversed hUC-MSCs-EVs effects on reducing LPS-induced neuronal apoptosis. CONCLUSION: hUC-MSCs-EVs activated the PI3K/AKT pathway by carrying miR-29b-3p into SCI neurons and silencing PTEN, thus reducing neuronal apoptosis.

Simultaneous optimization of ultrasound-assisted extraction for total flavonoid content and antioxidant activity of the tender stem of Triarrhena lutarioriparia using response surface methodology.

The asparagus of Triarrhena lutarioriparia (TL) is a popular vegetable with abundant chemical compounds in China. This study aims to optimize the ultrasound-assisted extraction (UAE) method for its content of total flavonoid and antioxidant activities by response surface methodology (RSM). Box-Behnken design was adopted to evaluate the influences of ethanol concentration, extraction time and solvent-to-sample ratio on the extraction yield of total flavonoid and the antioxidant activity. Considering the maximum content of extracted total flavonoids and antioxidant activity, the optimal extraction conditions were acquired with 70% (v/v) ethanol by UAE for 60 min at a solvent-to-sample ratio of 40 mL/g. The proportion of the extraction of total flavonoid was 15.88 mg/g and antioxidant activity reached 79.53%. The RSM would be recommended as an appropriate model for simultaneous optimization of the UAE conditions for the content of total flavonoid and the antioxidant activity of asparagus of TL.

Early surgical intervention for active thoracic spinal tuberculosis patients with paraparesis and paraplegia.

BACKGROUND: To explore the therapeutic effect of early surgical intervention for active thoracic spinal tuberculosis (TB) patients with paraparesis and paraplegia. METHODS: Data on 118 active thoracic spinal TB patients with paraparesis and paraplegia who had undergone surgery at an early stage (within three weeks of paraparesis and paraplegia) from January 2008 to December 2014 were retrospectively analyzed. The operation duration, blood loss, perioperative complication rate, VAS score, ASIA grade and NASCIS score of neurological status rating, Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP), kyphotic Cobb's angle, and duration of bone graft fusion were analyzed to evaluate the therapeutic effects of surgery. RESULTS: The mean operating time was 194.2 minutes, and the mean blood loss was 871.2 ml. The perioperative complication rate was 5.9 %. The mean preoperative VAS score was 5.3, which significantly decreased to 3.2 after the operation and continued decreasing to 1.1 at follow up (P<0.05). All cases achieved an increase of at least one ASIA grade after operation. The rate of full neurological recovery for paraplegia (ASIA grade A and B) was 18.0 % and was significantly lower than the rate (100 %) for paraparesis (ASIA grade C and D) (P<0.05). On the NASCIS scale, the difference in the neurological improvement rate between paraplegia (22.2 % ± 14.1 % in sensation and 52.2 % ± 25.8 % in movement) and paraparesis (26.7 % ± 7.5 % in sensation and 59.4 % ± 7.3 % in movement) was remarkable (P<0.05). Mean preoperative ESR and CRP were 73.1 mm /h and 82.4 mg/L, respectively, which showed a significant increase after operation (P>0.05), then gradually decreased to 11.5 ± 1.8 mm/h and 2.6 ± 0.82 mg/L, respectively, at final follow up (P<0.05). The mean preoperative kyphotic Cobb's angle was 21.9º, which significantly decreased to 6.5º after operation (P<0.05) while kyphotic correction was not lost during follow up (P>0.05). The mean duration of bone graft fusion was 8.6 ± 1.3 months. CONCLUSIONS: Early surgical intervention may be beneficial for active thoracic spinal TB patients with paraparesis and paraplegia, with surgical intervention being more beneficial for recovery from paraparesis than paraplegia.

Comparison of diagnostic efficacy of MRI and PET/CT in lung cancer of mouse with spinal metastasis.

This study aimed to compare the diagnostic efficacy of MRI and PET/CT in lung cancer of mouse with spinal metastasis. 40 healthy Balb/c nude mice were selected. 0.1 ml of human lung cancer cell A549 bacterial suspension was injected by the left ventricle injection method to establish a lung cancer spinal metastasis model, and the abnormal signs of the nude mice were closely observed. When the body weight was reduced by 20%, micro PET/CT imaging and small coil MRI imaging were applied after intraperitoneal injection of thiopental anesthesia in nude mice. After the imaging was completed, the nude mouse was dissected and the spinal tumor was removed. The nature of spinal metastases was diagnosed by the pathology department. 5 nude mice died of abdominal infection, 2 nude mice had no spinal tumors, and the remaining 33 nude mice were successfully modeled. 33 nude mice were confirmed by pathology to have 64 metastatic vertebral lesions, among them, there were 7 cervical vertebrae, 24 thoracic vertebrae, 16 lumbar vertebrae, 6 sacral vertebrae and 11 caudal vertebrae. The sensitivity of MRI in the diagnosis of spinal metastases was 78.13%, and specificity was 56.25%. The sensitivity of PET/CT for the diagnosis of spinal metastases was 92.19%, and specificity was 78.95%. The specificity and positive predictive value of PET/CT for the diagnosis of spinal metastases were not significantly different from those of MRI (P> 0.05). The sensitivity, accuracy and negative predictive values were significantly higher than those of MRI (P< 0.05). PET/CT is superior to MRI in the diagnosis of spinal metastases, and its sensitivity, accuracy and negative predictive values were significantly higher than those of MRI (P< 0.05). It is worthy to be further promoted in clinical practice.

Intramolecular C-H Functionalization Followed by a [2σ + 2π] Addition via an Intermediate Nickel-Nitridyl Complex.

Irradiation of a disphenoidal Ni(II) azido complex, [Cz tBu(Pyr iPr)2NiN3] (1), revealed an unprecedented nickel complex, [Cz tBu(Pyr iPr)(NH2-Pyr iPr)] (2), in >90% isolated yield. As evidenced by single-crystal X-ray diffraction, 2 is produced by double intramolecular C-H activation of a putative nickel-nitridyl intermediate, [Cz tBu(Pyr iPr)2Ni-⃛N•]. Calculations support the generation of an intermediate with significant nitridyl radical character after the loss of N2, which, in turn, undergoes tandem C-H activations, leading to functionalized intermediates and products. This is an unprecedented example of transient Ni-⃛N•-promoted intramolecular C-H functionalization, followed by a [2σ + 2π] addition, yielding bis-metallacyclic product 2. Complex 2 is also observed from the reaction of Ni(I) precursor Cz tBu(Pyr iPr)2Ni (3) and Me3SiN3, suggesting a unique thermal route toward a masked nickel-nitridyl intermediate.

Clinical Characteristics of 1378 Inpatients with Spinal Tuberculosis in General Hospitals in South-Central China.

In this retrospective study, charts of inpatients with spinal tuberculosis (STB) treated in large-scale general hospitals in Changsha, Hunan, China, between 2007 and 2016 were reviewed to investigate their clinical characteristics. Demographic, epidemiological and clinical features, imaging findings, treatment methods, and prognosis were summarized and analyzed. There were 1378 patients, 805 males and 573 females, with a mean age of 43.7 years. The mean interval between symptom onset and diagnosis was 16.0 months (range 15 days-240 months). The incidence of back pain, radicular pain and symptoms of systemic toxicity was 92.5%, 40.1%, and 32.1%, respectively. The rate of neurological impairment was 49.9 %. STB was present in two or more vertebrae in 91.1% of patients, with two adjacent vertebrae being involved in 67.9% of them. The lumbar segment (38.2%) was the most frequently affected, followed by the thoracic spine (35.7%). The sacrococcygeal area was the least frequently involved (0.8%). Abscesses were detected in 65.5% of patients. One thousand patients (72.6%) were managed with surgery and 378 (27.4%) with anti-TB drugs only. Cure was achieved in 1215 patients (88.2%), whereas 49 (3.5 %) had relapses. Concomitant pulmonary TB (PTB) was diagnosed in 366 patients (26.6%) and 63 (4.6%) had concomitant diabetes. Compared with the previous five years, the number of older patients, urban patients, and medical staff with STB had increased by 6.1%, 5.2%, and 1.3%, respectively in the five years studied. STB remains a severe public health problem that cannot be ignored. Most of the patients ignored early symptoms and therefore received untimely treatment. Thus, surveillance for and treatment of STB in South-central China requires strengthening. In addition to the current China-wide database of patients with PTB, a China-wide database of patients with STB should also be set up.

Osteogenic protein-1 attenuates nucleus pulposus cell apoptosis through activating the PI3K/Akt/mTOR pathway in a hyperosmotic culture.

BACKGROUND: Previous studies have indicated that osteogenic protein-1 has protective effects on the biological functions of intervertebral disc cells. Hyperosmolarity is an important physicochemical factor within the disc nucleus pulposus (NP) region, which obviously promotes NP cell apoptosis. OBJECTIVE: To study the effects of osteogenic protein-1 (OP-1) on NP cell apoptosis induced by hyperosmolarity and the potential signaling transduction pathway. METHODS: Rat NP cells were cultured in a hyperosmotic medium with or without OP-1 addition for 7 days. Inhibitor 294002 and inhibitor FK-506 were used to investigate the role of the PI3K/Akt/mTOR pathway in this process. NP cell apoptosis were evaluated by cell apoptosis ratio, activity of caspase-3/9 and gene/protein expression of apoptosis-related molecules (Bax, Bcl-2, caspase-3/cleaved caspase-3 and cleaved PARP). RESULTS: OP-1 addition obviously decreased cell apoptosis ratio and caspase-3/9 activity, down-regulated gene/protein expression of pro-apoptosis molecules (Bax, caspase-3/cleaved casepase-3 and cleaved PARP), up-regulated gene/protein expression of anti-apoptosis molecule (Bcl-2) in a hyperosmotic culture. Moreover, OP-1 addition significantly increased protein expression of p-Akt and p-mTOR. Further analysis showed that addition of LY294002 and FK-506 partly attenuated these protective effects of OP-1 against NP cell apoptosis and activation of the PI3K/Akt/mTOR pathway in a hyperosmotic culture. CONCLUSION: OP-1 can attenuate NP cell apoptosis through activating the PI3K/Akt/mTOR pathway in a hyperosmotic culture. The present study sheds a new light on the protective role of OP-1 in regulating disc cell biology and provides some theoretical basis for the application of OP-1 in retarding/regenerating disc degeneration.

Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA.

PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus-stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full-length cDNA (named CiPRKRA, KT891991) was cloned and identified. The full-length cDNA is comprised of a 5' UTR (36 bp), a 3' UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT-PCR showed that CiPRKRA was significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA in vivo. Recombinant plasmid of pGL3-PRKRAPro was constructed and transiently co-transfected into CIK cells with pcDNA3.1-CiIRF2 and pcDNA3.1-CiATF4, respectively. The results showed that both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription.

Ctenopharyngodon idella NF-κB subunit p65 modulates the transcription of IκBα in CIK cells.

NF-κB is an important transcription factor for regulating the multiple inflammatory and immune related gene transcription. It can bind with the nuclear factor κB site within the promoter of target genes to regulate their transcriptions. p65, the all-important subunit of NF-κB, is ubiquitously expressed in cells. In the present study, we cloned and identified the p65 subunit from grass carp (Ctenopharyngodon idella) (named Cip65) by homologous cloning and RACE technique. The full length of Cip65 cDNA is 2481 bp along with 9 bp 5' UTR, 639 bp 3' UTR and the largest open reading frame (1833 bp) encoding a polypeptide of 610 amino acids with a well conserved Rel-homology domain (RHD) in N-terminal and a putative transcription activation domain (TAD) in C-terminal. Cip65 gathers with other teleost p65 proteins to form a fish-specific clade clearly distinct from those of mammalian and amphibian counterparts on the phylogenetic tree. In CIK (C. idellus kidney) cells, the expression of Cip65 was significantly up-regulated under the stimulation with Poly I:C. As one member of the NF-κB inhibitor protein (IκB) family, IκBα can dominate the activity of NF-κB by interacting with it. To study the molecular mechanisms of negative feedback loop of NF-κB signaling in fish, we cloned grass carp IκBα (CiIκBα) promoter sequence. CiIκBα promoter is 414 bp in length containing two RelA binding sites and a putative atypical TATA-box. Meanwhile, Cip65 and its mutant proteins including C-terminus deletion mutant of Cip65 (Cip65-ΔC) and N-terminus deletion mutant of Cip65 (Cip65-ΔN) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, Cip65 rather than Cip65-ΔC and Cip65-ΔN showed high affinity with CiIκBα promoter sequence by gel mobility shift assays. In vivo, the cotransfection of pcDNA3.1-Cip65 (or pcDNA3.1-Cip65-ΔC, pcDNA3.1-Cip65-ΔN respectively) with pGL3-CiIκBα and pRL-TK renilla luciferase plasmid into CIK cells showed that pcDNA3.1-Cip65 rather than pcDNA3.1-Cip65-ΔC and pcDNA3.1-Cip65-ΔN, can increase the luciferase activity. Taken together, these results suggested that Cip65 can regulate the expression of CiIκBα and works as a negative feedback loop in NF-κB pathway.