The Prognostic Value of Alpha-Fetoprotein Ratio in Patients With Resectable Alpha-Fetoprotein-Negative Hepatocellular Carcinoma.

PURPOSE: This study aimed to investigate the prognostic value of alpha-fetoprotein (AFP) ratio in patients with AFP-negative hepatocellular carcinoma (HCC). PATIENTS AND METHODS: We retrospectively analyzed 600 AFP-negative HCC patients who underwent hepatectomy. The AFP ratio was calculated as the ratio of AFP level 1 week before surgery to the level 20-40 days after hepatectomy. Immunohistochemistry assay was used to assess protein expression in HCC tissue. The primary outcome measures were overall survival (OS) and disease-free survival (DFS). RESULTS: The study found that a cutoff value of 1.6 ng/ml for AFP ratio, determined using X-tile software, was optimal for predicting prognosis. Patients with a high AFP ratio had a worse prognosis compare to those with a low AFP ratio (DFS, P = .026; OS, P = .034). Patients with a high AFP ratio had a worse prognosis compared to those with a low AFP ratio. Multivariate analysis revealed that AFP ratio >1.6, negative HepPar-1 expression, and vascular invasion were independent predictors of both DFS and OS. Vascular invasion had a higher area under the curve (AUC) than AFP ratio and HepPar-1 expression in predicting recurrence and death. The combination of AFP ratio, HepPar-1 expression, and vascular invasion provided better predictive accuracy for DFS and OS. CONCLUSION: The AFP ratio is a potential prognostic marker for AFP-negative HCC patients after hepatectomy. Combining the analysis of AFP ratio with HepPar-1 expression and vascular invasion can enhance the accuracy of predicting prognosis in these patients.

Effect of individualized positive end-expiratory pressure based on electrical impedance tomography guidance on pulmonary ventilation distribution in patients who receive abdominal thermal perfusion chemotherapy.

Background: Electrical impedance tomography (EIT) has been shown to be useful in guiding individual positive end-expiratory pressure titration for patients with mechanical ventilation. However, the appropriate positive end-expiratory pressure (PEEP) level and whether the individualized PEEP needs to be adjusted during long-term surgery (>6 h) were unknown. Meanwhile, the effect of individualized PEEP on the distribution of pulmonary ventilation in patients who receive abdominal thermoperfusion chemotherapy is unknown. The primary aim of this study was to observe the effect of EIT-guided PEEP on the distribution of pulmonary ventilation in patients undergoing cytoreductive surgery (CRS) combined with hot intraperitoneal chemotherapy (HIPEC). The secondary aim was to analyze their effect on postoperative pulmonary complications. Methods: A total of 48 patients were recruited and randomly divided into two groups, with 24 patients in each group. For the control group (group A), PEEP was set at 5 cm H2O, while in the EIT group (group B), individual PEEP was titrated and adjusted every 2 h with EIT guidance. Ventilation distribution, respiratory/circulation parameters, and PPC incidence were compared between the two groups. Results: The average individualized PEEP was 10.3 ± 1.5 cm H2O, 10.2 ± 1.6 cm H2O, 10.1 ± 1.8 cm H2O, and 9.7 ± 2.1 cm H2O at 5 min, 2 h, 4 h, and 6 h after tracheal intubation during CRS + HIPEC. Individualized PEEP was correlated with ventilation distribution in the regions of interest (ROI) 1 and ROI 3 at 4 h mechanical ventilation and ROI 1 at 6 h mechanical ventilation. The ventilation distribution under individualized PEEP was back-shifted for 6 h but moved to the control group's ventral side under PEEP 5 cm H2O. The respiratory and circulatory function indicators were both acceptable either under individualized PEEP or PEEP 5 cm H2O. The incidence of total PPCs was significantly lower under individualized PEEP (66.7%) than PEEP 5 cm H2O (37.5%) for patients with CRS + HIPEC. Conclusion: The appropriate individualized PEEP was stable at approximately 10 cm H2O during 6 h for patients with CRS + HIPEC, along with better ventilation distribution and a lower total PPC incidence than the fixed PEEP of 5 cm H2O.Clinical trial registration: identifier ChiCTR1900023897.

Lactate Improves Long-term Cognitive Impairment Induced By Repeated Neonatal Sevoflurane Exposures Through SIRT1-mediated Regulation of Adult Hippocampal Neurogenesis and Synaptic Plasticity in Male Mice.

Repeated neonatal exposures to sevoflurane induce long-term cognitive impairment that has been reported to have sex-dependent differences. Exercise promotes learning and memory by releasing lactate from the muscle. The study tested the hypothesis that lactate may improve long-term cognitive impairment induced by repeated neonatal exposures to sevoflurane through SIRT1-mediated regulation of adult hippocampal neurogenesis and synaptic plasticity. C57BL/6 mice of both genders were exposed to 3% sevoflurane for 2 h daily from postnatal day 6 (P6) to P8. In the intervention experiments, mice received lactate at 1 g/kg intraperitoneally once daily from P21 to P41. Behavioral tests including open field (OF), object location (OL), novel object recognition (NOR), and fear conditioning (FC) tests were performed to assess cognitive function. The number of 5-Bromo-2'- deoxyuridine positive (BrdU+) cells and BrdU+/DCX+ (doublecortin) co-labeled cells, expressions of brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeletal-associated protein (Arc), early growth response 1 (Egr-1), SIRT1, PGC-1α and FNDC5, and long-term potentiation (LTP) were evaluated in the hippocampus. Repeated exposures to sevoflurane induced deficits in OL, NOR and contextual FC tests in male but not female mice. Similarly, adult hippocampal neurogenesis, synaptic plasticity-related proteins and hippocampal LTP were impaired after repeated exposures to sevoflurane in male but not female mice, which could rescue by lactate treatment. Our study suggests that repeated neonatal exposures to sevoflurane inhibit adult hippocampal neurogenesis and induce defects of synaptic plasticity in male but not female mice, which may contribute to long-term cognitive impairment. Lactate treatment rescues these abnormalities through activation of SIRT1.

Polyphenol-driven facile assembly of a nanosized acid fibroblast growth factor-containing coacervate accelerates the healing of diabetic wounds.

Diabetic wounds are challenging to heal due to complex pathogenic abnormalities. Routine treatment with acid fibroblast growth factor (aFGF) is widely used for diabetic wounds but hardly offers a satisfying outcome due to its instability. Despite the emergence of various nanoparticle-based protein delivery approaches, it remains challenging to engineer a versatile delivery system capable of enhancing protein stability without the need for complex preparation. Herein, a polyphenol-driven facile assembly of nanosized coacervates (AE-NPs) composed of aFGF and Epigallocatechin-3-gallate (EGCG) was constructed and applied in the healing of diabetic wounds. First, the binding patterns of EGCG and aFGF were predicted by molecular docking analysis. Then, the characterizations demonstrated that AE-NPs displayed higher stability in hostile conditions than free aFGF by enhancing the binding activity of aFGF to cell surface receptors. Meanwhile, the AE-NPs also had a powerful ability to scavenge reactive oxygen species (ROS) and promote angiogenesis, which significantly accelerated full-thickness excisional wound healing in diabetic mice. Besides, the AE-NPs suppressed the early scar formation by improving collagen remodeling and the mechanism was associated with the TGF-ß/Smad signaling pathway. Conclusively, AE-NPs might be a potential and facile strategy for stabilizing protein drugs and achieving the scar-free healing of diabetic wounds. STATEMENT OF SIGNIFICANCE: Diabetic chronic wound is among the serious complications of diabetes that eventually cause the amputation of limbs. Herein, a polyphenol-driven facile assembly of nanosized coacervates (AE-NPs) composed of aFGF and EGCG was constructed. The EGCG not only acted as a carrier but also possessed a therapeutic effect of ROS scavenging. The AE-NPs enhanced the binding activity of aFGF to cell surface receptors on the cell surface, which improved the stability of aFGF in hostile conditions. Moreover, AE-NPs significantly accelerated wound healing and improved collagen remodeling by regulating the TGF-ß/Smad signaling pathway. Our results bring new insights into the field of polyphenol-containing nanoparticles, showing their potential as drug delivery systems of macromolecules to treat diabetic wounds.

Alpha-Fetoprotein Ratio Predicts Alpha-Fetoprotein Positive Hepatocellular Cancer Patient Prognosis after Hepatectomy.

BACKGROUND: This study was conducted to investigate the effect of alpha-fetoprotein (AFP) ratio on the prognosis of AFP-positive hepatocellular carcinoma (HCC) patients after hepatectomy. METHODS: We retrospectively included 879 HCC patients with AFP-positive who underwent hepatectomy from February 2012 to October 2017 and randomly divided into training cohort and validation cohort. AFP ratio was equal to the AFP level within one week before hepatectomy to AFP level within 20-40 days after surgery. The end point of follow-up was disease-free survival (DFS) and overall survival (OS). RESULTS: AFP ratio was not associated with clinical characteristics in training cohort and validation cohort. According to the X-tile software, the optimum cut-off point was 17.8 for AFP ratio. Significant differences between AFP ratio high and AFP ratio low were observed in DFS and OS in both cohort (p < 0.05). Kaplan-Meier curves and receiver-operating curves were showed that AFP ratio was better than AFP level preoperation in predicting the prognosis of AFP-positive HCC patients after hepatectomy. The multivariate analysis demonstrated that AFP ratio was a significant independent risk factor for both OS and DFS in HCC patients with AFP-positive. CONCLUSIONS: AFP ratio might be a prognosis predictor for HCC patients with AFP-positive after hepatectomy.

Cannabinoid receptors promote chronic intermittent hypoxia-induced breast cancer metastasis via IGF-1R/AKT/GSK-3ß.

The progression of breast cancer is closely related to obstructive sleep apnea-hypopnea syndrome (OSAHS). Low concentrations of cannabinoids promote tumor proliferation. However, the role of cannabinoid receptors (CBs) in chronic intermittent hypoxia (CIH)-induced breast cancer has not been reported. The migration and invasion of breast cancer cell lines (MCF-7 and T47D) were measured by scratch assay and transwell assay. Gene and protein expressions were analyzed by qPCR and western blotting. Tumor xenograft mice model were established to evaluate the function of CBs. We observed that chronic hypoxia (CH) and CIH increased CBs expression and promoted migration and invasion in breast cancer. Mice grafted with MCF-7 exhibited obvious tumor growth, angiogenesis, and lung metastasis in CIH compared with CH and control. In addition, CIH induced CBs expression, which subsequently activated insulin-like growth factor-1 receptor (IGF-1R)/AKT/glycogen synthase kinase-3ß (GSK-3ß) axis. Knockdown of CBs alleviated CIH-induced migration and invasion of breast cancer in vitro. Furthermore, CIH exaggerated the malignancy of breast cancer and silencing of CBs suppressed tumor growth and metastasis in vivo. Our study contributed to understanding the role of CIH in breast cancer development modulation.

Smoking status combined with tumor mutational burden as a prognosis predictor for combination immune checkpoint inhibitor therapy in non-small cell lung cancer.

BACKGROUND: This study aimed to explore the prognostic value of tumor mutational burden (TMB) combined with smoking status in advanced non-small cell lung cancer (NSCLC) patients who received immune checkpoint inhibitor therapy (anti PD-1/PD-L1 therapy) combined with chemotherapy or anti-angiogenesis therapy. METHODS: We conducted a retrospective analysis of NSCLC patients who underwent next-generation sequencing test (either 295-gene panel NGS or 1021-gene panel NGS) from September 2017 to November 2020. The relationship between TMB and smoking status was investigated. Kaplan-Meier survival analysis was used to compare progression-free survival (PFS) of the NSCLC patients who received combination immunotherapy grouped by TMB value and smoking status. RESULTS: We enrolled 323 cases and 388 cases of NSCLC patients in the 295-gene panel cohort and 1021-gene panel cohort, respectively. Positive correlation between TMB and smoking status was found in lung adenocarcinoma, but not in lung squamous cell carcinoma. Participants with both high TMB and smoking status who received immune checkpoint therapy combined with chemotherapy or anti-angiogenesis therapy had longer PFS than other participants (p < 0.05). CONCLUSIONS: The combination of TMB with smoking status might be a potential predictor for the efficacy of combination immunotherapy in advanced NSCLC.

RIP1 promotes proliferation through G2/M checkpoint progression and mediates cisplatin-induced apoptosis and necroptosis in human ovarian cancer cells.

Receptor-interacting protein 1 (RIP1, also known as RIPK1) is not only a tumor-promoting factor in several cancers but also mediates either apoptosis or necroptosis in certain circumstances. In this study we investigated what role RIP1 plays in human ovarian cancer cells. We showed that knockout (KO) of RIP1 substantially suppressed cell proliferation, accompanied by the G2/M checkpoint arrest in two human ovarian cancer cell lines SKOV3 and A2780. On the other hand, RIP1 KO remarkably attenuated cisplatin-induced cytotoxicity, which was associated with reduction of the apoptosis markers PARP cleavage and the necroptosis marker phospho-MLKL. We found that RIP1 KO suppressed cisplatin-induced ROS accumulation in both SKOV3 and A2780 cells. ROS scavenger BHA, apoptosis inhibitor Z-VAD or necroptosis inhibitor NSA could effectively suppress cisplatin's cytotoxicity in the control cells, suggesting that ROS-mediated apoptosis and necroptosis were involved in cisplatin-induced cell death. In addition, blocking necroptosis with MLKL siRNA effectively attenuated cisplatin-induced cytotoxicity. In human ovarian cancer A2780 cell line xenograft nude mice, RIP1 KO not only significantly suppressed the tumor growth but also greatly attenuated cisplatin's anticancer activity. Our results demonstrate a dual role of RIP1 in human ovarian cancer: it acts as either a tumor-promoting factor to promote cancer cell proliferation or a tumor-suppressing factor to facilitate anticancer effects of chemotherapeutics such as cisplatin.

Obstructive sleep apnea syndrome and causal relationship with female breast cancer: a mendelian randomization study.

Although observational studies have reported a positive association between obstructive sleep apnea syndrome (OSAS) and breast cancer (BC) risk, causality remains inconclusive. We aim to explore whether OSAS is associated with etiology of BC by conducting a two-sample Mendelian randomization (MR) study in a Chinese population and Asian population from the Breast Cancer Association Consortium (BCAC). We found a detrimental causal effect of OSAS on BC risk in the primary analysis of our samples (IVW OR, 2.47 for BC risk per log-odds increment in OSAS risk, 95% CI = 1.86-3.27; P = 3.6×10-10). This was very similar to results of the direct observational case-control study between OSAS and BC risk (OR = 2.80; 95% CI = 2.24-3.50; P =1.4×10-19). Replication in the Asian population of the BCAC study also supported our results (IVW OR, 1.33 for BC risk per log-odds increment in OSAS risk, 95% CI = 1.13-1.56; P = 0.0006). Sensitivity analyses confirmed the robustness of our findings. We provide novel evidence that genetically determined higher risk of OSAS has a causal effect on higher risk of BC. Further studies focused on the mechanisms of the relationship between OSAS and breast carcinogenesis are needed.

Overinhibition mediated by parvalbumin interneurons might contribute to depression-like behavior and working memory impairment induced by lipopolysaccharide challenge.

Systemic inflammation induces cognitive impairments via unclear mechanisms. Accumulating evidence has demonstrated that a subset of neurons that express parvalbumin (PV) play a critical role in regulation of cognitive and emotional behavior. Thus, the aim of the present study was to test whether disruption of PV interneuron mediates systemic inflammation-induced depression-like behavior and working memory impairment by lipopolysaccharide (LPS) challenge. Here we showed that LPS induces depression-like behavior and working memory impairment, coinciding with increased PV expression, enhanced GABAergic transmission, and impaired long-term potentiation (LTP) in the hippocampus. Notably, systemic administration of NMDA (N-methyl-D-aspartate) receptor (NMDAR) antagonist ketamine was able to interfere with PV expression and reverse depression-like behavior and working memory impairment, which is probably mediated by reversing impaired LTP. In addition, flumazenil, a competitive antagonist acting at the benzodiazepine binding site of the GABAA receptor, also ameliorated these abnormal behaviors. Collectively, our study added growing evidence to the limited studies that overinhibition mediated by PV interneurons might play a critical role in LPS-induced depression-like behavior and working memory impairment.

CB1 receptor antagonist rimonabant protects against chronic intermittent hypoxia-induced bone metabolism disorder and destruction in rats.

OBJECTIVE: The endocannabinoid system (ECS) regulates bone turn-over and remodeling. Chronic intermittent hypoxia (CIH) occurring during obstructive sleep apnea (OSA) may lead to disorders of the ECS and bone metabolism abnormalities. This study aimed to investigate whether or not the cannabinoid receptor 1 (CB1R) antagonist rimonabant (Ri) alleviates bone metabolism abnormalities and bone destruction induced by chronic intermittent hypoxia (CIH). METHODS: Healthy male Sprague Dawley (SD) rats (n=48) were randomly divided into 6 groups of 8 rats: 2 normal control (NC) groups, 2 intermittent hypoxia (IH) groups, and 2 IH + Ri groups. Rats in NC groups breathed room air for 4 weeks (4w NC group) and 6 weeks (6w NC group). Rats in IH groups experienced IH environment for 4 weeks (4w IH group) and 6 weeks (6w IH group). In addition to the same IH exposure, rats in IH + Ri group were given daily intraperitoneal injection of Ri at the dosage of 1.5 mg/kg/d for 4 weeks (4w IH + Ri group) and 6 weeks (6w IH + Ri group). Levels of serum tartrate-resistant acid phosphatase (TRAP, a marker of bone resorption) were determined by ELISA. Hematoxylin and eosin (HE) staining was performed on bone sections to observe the changes in bone microstructure. Expression of CB1R in bone tissue was determined by immunohistochemistry. RESULTS: TRAP levels were higher in the 4w IH and 6w IH groups than in the 4w NC and 6w NC groups; TRAP levels were lower in the 4w IH + Ri and 6w IH + Ri groups than in the 4w IH and 6w IH groups. HE staining showed that the morphology of bone cells in the NC group was normal, but the 4w IH group had mild edema of bone cells, reduction in trabecular bone, and destruction of bone microstructure. Changes were more severe in the 6w IH group than 4w IH. The 4w IH + Ri group was slightly improved compared with the 4w IH group. The 6w IH + Ri group was improved compared with the 4w IH + Ri group. The results of immunohistochemistry showed that the expression of CB1R in IH group was significantly higher than that in NC group. The expression of CB1R in the IH + Ri group was lower than that in the IH group. With the prolongation of hypoxia, the expression of CB1R in bone cells of IH group increased. The expression level of CB1R in IH + Ri group decreased with the prolongation of intervention time. Correlation analysis showed that the expression rate of CB1R in bone cells was positively correlated with the level of TRAP in serum. CONCLUSION: CIH increases serum TRAP levels and triggers metabolic bone disorder by activating bone CB1R. Intervention with CB1R antagonist (rimonabant) reduces the bone dysmetabolism in the CIH rat model.

Mixed adenoneuroendocrine carcinoma of the liver: A rare case report.

A 55-year-old woman presented with chest and back pain of unknown cause. Contrast-enhanced computed tomography revealed two low-density tumors, sized 4.6 and 4.4 cm, in the hepatic caudate and left inner lobes, respectively. There are multiple enlarged lymph nodes around the abdominal aorta, hepatogastric ligament and gastrosplenic ligament. At the same time, there were multiple enlarged lymph nodes between the portal vein and the vena cava. Upper gastrointestinal endoscopy revealed chronic non-atrophic gastritis and esophagitis (grade B). Endoscopic examination of the lower digestive tract revealed polyps of the colon, diagnosed as tubular adenomas following biopsy and histopathological examination. The patient underwent left three hepatic resection (including left inner lobe, left outer lobe and right anterior lobe resection), abdominal lymph node dissection, right liver tumor radiofrequency ablation, hepatic caudate lobe resection, intestinal adhesion release, vena cava formation, portal vein repair and hilar cholangioplasty. The pathological examination of the resected specimens revealed intrahepatic bile duct carcinoma and hepatic parenchymal neuroendocrine tumor (NET). In addition, liver solid portions consisted of tumor cells with characteristic salt-and-pepper nuclei. Immunohistochemical examination revealed expression of the neuroendocrine marker synaptophysin in this solid component, confirming the diagnosis of NET. Furthermore, the MIB-1 proliferation index of the NET was higher compared with that of the adenocarcinoma, and lymph node invasion by the NET component was detected, indicating a neuroendocrine carcinoma (NEC, or NET G3). The diagnosis of mixed adenoneuroendocrine carcinoma of the liver was confirmed based on the World Health Organization 2010 criteria. Taking into consideration the patient's poor general condition, only symptomatic supportive treatment was administered postoperatively, without chemotherapy. Contrast-enhanced computed tomography at 45 days postoperatively revealed disease progression, with metastases in the liver stump, abdominal lymph nodes, spine and pelvis. The patient remained on symptomatic supportive treatment and succumbed to disease progression 3 months after surgery.

Intergenerational Effects of Sevoflurane in Young Adult Rats.

BACKGROUND: Sevoflurane administered to neonatal rats induces neurobehavioral abnormalities and epigenetic reprogramming of their germ cells; the latter can pass adverse effects of sevoflurane to future offspring. As germ cells are susceptible to reprogramming by environmental factors across the lifespan, the authors hypothesized that sevoflurane administered to adult rats could induce neurobehavioral abnormalities in future offspring, but not in the exposed rats themselves. METHODS: Sprague-Dawley rats were anesthetized with 2.1% sevoflurane for 3 h every other day between postnatal days 56 and 60. Twenty-five days later, exposed rats and nonexposed controls were mated to produce offspring. RESULTS: Adult male but not female offspring of exposed parents of either sex exhibited deficiencies in elevated plus maze (mean ± SD, offspring of both exposed parents vs. offspring of control parents, 35 ± 12 vs. 15 ± 15 s, P < 0.001) and prepulse inhibition of acoustic startle (offspring of both exposed parents vs. offspring of control parents, 46.504 ± 13.448 vs. 25.838 ± 22.866%, P = 0.009), and increased methylation and reduced expression of the potassium ion-chloride ion cotransporter KCC2 gene (Kcc2) in the hypothalamus. Kcc2 was also hypermethylated in sperm and ovary of the exposed rats. Surprisingly, exposed male rats also exhibited long-term abnormalities in functioning of the hypothalamic-pituitary-gonadal and -adrenal axes, reduced expression of hypothalamic and hippocampal Kcc2, and deficiencies in elevated plus maze (sevoflurane vs. control, 40 ± 24 vs. 25 ± 12 s, P = 0.038) and prepulse inhibition of startle (sevoflurane vs. control, 39.905 ± 21.507 vs. 29.193 ± 24.263%, P < 0.050). CONCLUSIONS: Adult sevoflurane exposure affects brain development in male offspring by epigenetically reprogramming both parental germ cells, while it induces neuroendocrine and behavioral abnormalities only in exposed males. Sex steroids may be required for mediation of the adverse effects of adult sevoflurane in exposed males.

[Electroacupuncture of Multiple Acupoints Is Significantly Superior to That of Single Acupoint in Improving Sleep by Regulating Serum Sleep-promoting and Awakening-promoting Factors in Insomnia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of single and multiple acupoints on sleep and concentrations of interlukin-1 ß(IL-1 ß), brain-derived neurotrophic factor (BDNF), prostaglandin D2(PGD2) and melatonin (MLT, sleep-promoting factors) and corticosterone (CORT, awakening-promoting factor) in the serum in insomnia rats, so as to explore its efficacy difference and the mechanism underlying improving sleep. METHODS: Fifty-four male SD rats were randomly divided into control, model, EA-Baihui (GV 20), EA-Shenmen (HT 7), EA-Sanyinjiao (SP 6) and EA-GV 20＋HT 7＋SP 6 groups (n＝9 rats in each group). The insomnia model was established by intraperitoneal injection of para-chlorophenylalanine (PCPA, 300 mg/kg) once daily for 2 days. In the EA-GV 20, EA-HT 7, EA-SP 6 and EA-GV 20＋HT 7＋SP 6 groups, EA stimulation was administrated for 30 min, once a day for 4 days. The sleep onset latency and sleep duration were measured after intraperitoneal injection of pentobarbital sodium (35 mg/kg). The concentrations of IL-1 ß， BDNF, MLT, PGD2and CORT in the serum were detected by ELISA. RESULTS: After EA stimulation of GV 20, HT 7, SP 6 and GV 20＋HT 7＋SP 6, the sleep latency was significantly shortened (P<0.05, P<0.01, except SP 6), and the sleep duration was remarkably prolonged in comparison with the model group (P<0.05, P<0.01), and the therapeutic effects of EA-GV 20＋HT 7＋SP 6 were significantly superior to those of EA-GV 20, EA-HT 7 and EA-SP 6 in shortening the sleep latency and lengthening the sleep duration (P<0.05). Following modeling, the concentrations of IL-1 ß， BDNF, PGD2 and MLT were significantly down-regulated, and the CORT level was markedly up-regulated in the model group relevant to the control group (P<0.05). Following EA，modeling induced dramatic decrease of serum IL-1 ß， BDNF, PGD2 and MLT was considerably up-regulated, and the increased CORT level markedly down-regulated in the EA-GV 20, EA-HT 7, EA-SP 6 and EA-GV 20＋HT 7＋SP 6 groups (P<0.05). The effects of EA-GV 20＋HT 7＋SP 6 were evidently superior to those of EA-GV 20 and EA-SP 6 in up-regulating serum IL-1 ß， BDNF and PGD2levels, and to those of HT 7, GV 20 and SP 6 in up-regulating serum MLT level, and significantly superior to those of EA-ST 7 and EA-SP 6 in down-regulating serum CORT (P<0.05). CONCLUSION: EA stimulation of HT 7, GV 20, SP 6 and GV 20＋HT 7＋ SP 6 can significantly improve the sleep in insomnia rats, which is closely associated with its effects in regulating serum sleep-promoting factors and awakening-promoting factor. Joint administration of EA of GV 20＋HT 7＋ SP 6 has a better effect than the single acupoint mentioned above.

Toll-Like Receptor 4 (TLR-4) Pathway Promotes Pulmonary Inflammation in Chronic Intermittent Hypoxia-Induced Obstructive Sleep Apnea.

BACKGROUND Studies have shown that intermittent hypoxia mimics obstructive sleep apnea in causing pulmonary inflammation, but the mechanism is not yet clear.TLR-4 is a recognized proinflammatory factor, so the purpose of this study was to assess the function of TLR-4 in pulmonary inflammation induced by chronic intermittent hypoxia simulating obstructive sleep apnea. MATERIAL AND METHODS Healthy male Wistar rats were divided into 3 groups (8 in each group): the normoxia control group (CG), the intermittent hypoxia group (IH), and the TLR4 antagonist TAK242 treatment group (3 mg/kg, daily), with exposure durations of 12 weeks and 16 weeks (HI). The morphological changes of lung tissue were determined with hematoxylin-eosin (HE) staining. The expressions of the TLR-4 pathway in lung tissue were tested by Western blotting and RT-PCR. The levels of IL-6 and TNF-a in serum and lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). The levels of SOD and MDA in lung tissue were detected by use of SOD and MDA kits, respectively. RESULTS After TAK242 treatment, damage to lung tissue was increased, and the expressions of TLR-4, MYD88, P65, IL-6, TNF-α, MDA, and SOD were decreased. Intermittent hypoxic exposure caused alveolar expansion, thickening of alveolar septum, and fusion of adjacent alveoli into larger cysts under intermittent hypoxia in a time-dependent manner. Compared with the CG and HI groups, the mean lining interval (MLI) become more thickened and the alveolar destruction index (DI) increased significantly in the IH group. CONCLUSIONS Chronic intermittent hypoxia causes pulmonary inflammatory response and the inflammatory pathway involved in TLR4 receptor may be one of the mechanisms that trigger lung inflammation.

Bone Metabolic Markers in Patients with Obstructive Sleep Apnea Syndrome.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is prevalent in obesity and is associated with many metabolic abnormalities. The relationship between OSAS and bone metabolism is still unclear. The aim of this study was to investigate the relationship between the severity of OSAS and bone metabolic markers. METHODS: A total of 119 obese males were enrolled in this study in spring months from 2015 to 2017. All candidates underwent polysomnography, and their bone mineral density (BMD) and the serum levels of total procollagen type 1 N-terminal propeptide (t-P1NP), N-terminal midfragment of osteocalcin (N-MID), ß-C-terminal telopeptide of type 1 collagen (ß-CTX), vitamin D (VD), and parathyroid hormone (PTH) were measured. The analysis of variance and Pearson correlation analysis were performed for data analyses. RESULTS: No significant differences in the mean values of BMD were observed among the obesity, mild-to-moderate OSAS, and severe OSAS groups; and the serum levels of t-P1NP and ß-CTX in the severe OSAS group were significantly higher than those in the obesity group (48.42 ± 23.78 ng/ml vs. 31.98 ± 9.85 ng/ml, P < 0.001; 0.53 ± 0.24 ng/ml vs. 0.41 ± 0.13 ng/ml, P = 0.011, respectively). The serum level of VD in the obesity group was significantly higher than those in the mild-to-moderate and severe OSAS groups (both P < 0.001), and decreased as the severity of OSAS increased (P < 0.001). The serum level of PTH in the severe OSAS group was significantly higher than those in the obesity and mild-to-moderate OSAS groups (both P < 0.001). The results of correlation analysis indicated that the level of apnea-hypopnea index (AHI) was correlated with the levels of t-P1NP (r = 0.396, P < 0.001), VD (r = -0.404, P < 0.001), and PTH (r = 0.400, P < 0.001), whereas the level of minimum O2saturation (SaO2min) was correlated with the levels of VD (r = 0.258, P = 0.016) and PTH (r = -0.376, P < 0.001). CONCLUSIONS: The levels of bone resorption and formation markers in patients with severe OSAS were significantly increased compared to obese men, and the severity of OSAS was correlated with the serum levels of t-P1NP, VD, and PTH.

Effects of RNA interference-mediated silencing of toll-like receptor 4 gene on proliferation and apoptosis of human breast cancer MCF-7 and MDA-MB-231 cells: An in vitro study.

Breast cancer is known as the most prevalent cancer in women worldwide, and has an undeniable negative impact on public health, both physically, and mentally. This study aims to investigate the effects of toll-like receptor 4 (TLR4) gene silencing on proliferation and apoptosis of human breast cancer cells to explore for a new theoretical basis for its treatment. TLR4 small interference RNA (siRNA) fragment recombinant plasmids were constructed, including TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3. Human breast cancer MCF-7 and MDA-MB-231 cells were assigned into blank, negative control (NC), TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3 groups. MCF-7 and MDA-MB-231 cell growth was detected by MTT assay. Apoptosis and cell cycle were determined by flow cytometry. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were conducted to determine the expression of TLR4, CDK4, cyclin D1, Livin, Bcl-2, p53, c-FLIP, and caspase-3. In comparison with the NC and blank groups, the TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3 groups showed decreased the expression of TLR4, inhibited proliferation of MCF-7 and MDA-MB-231 cells and promoted MCF-7 and MDA-MB-231 cell apoptosis, and the cells were blocked in G1 phase. In comparison with the NC and blank groups, in the TLR4 siRNA-1, TLR4 siRNA-2, and TLR4 siRNA-3 groups, siRNA-TLR4 significantly increased expression of p53 and caspase-3 in MCF-7 and MDA-MB-231 cells, while it decreased the expressions of CDK4, cyclinD1, Livin, Bal-2, and c-FLIP. The study demonstrates that TLR4 gene silencing inhibits proliferation and induces apoptosis of MCF-7 and MDA-MB-231 cells.

Upregulation of microRNA-335-5p reduces inflammatory responses by inhibiting FASN through the activation of AMPK/ULK1 signaling pathway in a septic mouse model.

Sepsis, as a systemic inflammatory response syndrome (SIRS) subtype, is generally characterized by infection. Emerging evidence has highlighted dysregulated microRNAs (miRNAs) are involved in the progression of sepsis. The aim of the study was to investigate the effects of miR-335-5p on inflammatory responses in a septic mouse model. The hypothesis was subsequently asserted that the FASN gene and AMPK/ULK1 signaling pathway may participate in the regulation of miR-335-5p. A septic mouse model was established in order to validate the effect of miR-335-5p on the inflammatory response by means of suppressing the endogenous expression of FASN by siRNA against FASN in endothelial cells. A target prediction program and luciferase activity was employed to ascertain as to whether miR--335-5p targets FASN. The levels of inflammatory factors including IL-6 and IL-1ß were determined by means of ELISA assay. RT-qPCR and western blot analysis were used to determine the AMPK/ULK1 signaling pathway-, apoptosis- and autophagy-related genes. Flow cytometry was employed in order to evaluate sepsis-induced cell apoptosis in response to miR-335-5p and FASN alternations. FASN was identified as a target gene of miR--335-5p. Gain- and loss-of-function studies revealed that miR-335-5p acted to enhance autophagy, reduce cell apoptosis, promote cell cycle entry in endothelial cells, and reduce inflammatory response through the modulation of pro- and anti-apoptotic factors in endothelial cells. The effect of miR-335-5p on endothelial cells was increased when FASN was suppressed by siRNA as well as when the AMPK/ULK1 signaling pathway was activated, suggesting that miR-335-5p influences sepsis by targeting and inhibiting FASN, and activating the AMPK/ULK1 signaling pathway. Our study provides evidence indicating that overexpressed miR-335-5p enhances autophagy by targeting FASN through activation of the AMPK/ULK1 signaling pathway working to alleviate the inflammatory response in septic mouse models, emphasizing the value of the functional upregulation of miR-335-5p as therapeutic strategy for sepsis.

[Transcutaneous Electrostimulation of Auricular Otopoints Reduces Epileptic Attack Possibly by Suppressing Hippocampal Gliocyte Proliferation and Regulating IL-6 and IL-10 Expression in Chronic Temporal Lobe Epilepsy Rats].

OBJECTIVE: To observe the effect of transcutaneous otopoint electrostimulaiton (TCOES) on seizure frequency, immunoreactivity of hippocampal gliocytes and expression of proinflammatory cytokine interleukin-6(IL-6) and anti-inflammatory cytokine IL-10 in chronic temporal lobe epilepsy (CTLE) rats, so as to investigate its antiepileptic mechanism. METHODS: Thirty-six SD rats were randomly divided into control, model and TCOES groups (n=12 in each group). The CTLE model was established by intraperitoneal injection (i.p.i.) of lithium chloride (127.2 mg/kg), scopolamine (1 mg/kg, 20 h after the 1st injection) and pilocarpine (10 mg/kg, 30 min after scopolamine injection). Rats of the control group were treated by i.p.i. of normal saline. TCOES (1 mA, 20 Hz) was applied to bilateral otopoint "Heart"-"Lung"-"Subcortex" region for 20 min, once daily for 6 weeks. The epileptic attack was observed by a video monitoring system. The numbers of ionized calcium-binding adapter molecule-1 (Iba 1)-labeled microgliacytes and glial fibrillary acidic protein (GFAP)-labeled astrocytes in the CA 1 and CA 3 regions of hippocampus were counted under light microscope after immunostaining, and the expression levels of hippocampal IL-6 and IL-10 proteins and genes were determined by immunofluorescence and quantitative real-time PCR, respectively. RESULTS: After TCOES intervention, the seizure frequency was significantly decreased in comparison with pre-treatment(P<0.05), modeling-induced dramatic increase of the numbers of microgliacytes and astrocytes,IL-6 immunoactivity in the hippocampal CA 1 and CA 3 regions, and IL-6 mRNA expression in the hippocampus were significantly suppressed (P<0.05), and hippo-campal IL-10 immunoactivity and mRNA expression were considerably up-regulated in comparison with the model group (P<0.05). CONCLUSIONS: TCOES intervention has an antiepileptic effect in CTLE rats, which may be associated with its effects in suppressing gliocyte proliferation, suppressing the expression of proinflammatory cytokine IL-6, and up-regulaiting the expression of anti-inflammatory cytokine IL-10 in the hippocampus.

[Effects of Electroacupuncture on Activities of Satellite Glial Cells of Dorsal Root Ganglia in Rats with Neck Incision Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Futu"(LI 18), etc. on activities of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) in rats with neck-incision pain so as to explore its mechanism underlying reduction of post-surgical pain of thyroidectomy. METHODS: Male SD rats were randomly divided into control, model, EA-Futu (LI 18), EA-Hegu (LI 4)-Neiguan (PC 6), and EA-Zusanli (ST 36)-Yanglingquan (GB 34) groups, with 20 rats in each group. The neck-incision pain model was established by making a longitudinal incision and repeated mechanical stimulation. In the EA-LI 18, EA-LI 4-PC 6 and EA-ST 36-GB 34 groups, EA stimulation was administrated for 30 min, once a day,continuously for 3 days. The thermal pain threshold (PT) of the neck-incision region was detected. The immunoactivity of glial fibrillary acidic protein (GFAP,a specific marker for SGCs) and connexin 43 (Cx 43) of DRGs (C 2-C 6) was determined by fluorescent immunohistochemistry. The expression levels of GFAP, IL-1 ß, IL-6, and TNF-α mRNAs were determined by quantitative Real-time PCR, and the contents of IL-1 ß,IL-6,TNF-α assayed by enzyme linked immunosorbent assay (ELISA) and the expression of Cx 43 protein was detected by Western blot. RESULTS: After EA intervention at LI 18 and LI 4-PC 6 (but not ST 36-GB 34), neck incision-induced reduction of the thermal PT was obviously prolonged in comparison with the model group (P<0.05),suggesting a pain relief. The expression levels of GFAP, IL-1 ß, IL-6 and TNF-α mRNAs and Cx 43 protein, and the contents of IL-1 ß, IL-6 and TNF-α in C 2-C 6 DRGs were all significantly up-regulated in the model group relevant to those of the control group (P<0.05). Following EA, modeling induced dramatic increase of expression of GFAP, IL-1 ß, IL-6 and TNF-α mRNAs and Cx 43 protein in both EA-LI 18 and EA-LI 4-PC 6 groups, and the contents of IL-1 ß and TNF-α in the EA-LI 18 group, IL-6 in the EA-LI 4-PC 6 group was considerably down-regulated (P<0.05). In comparison with the model group, no significant changes were found in all the abovementioned indexes of EA-ST 36 -GB 34 group except the down-regulated IL-1 ß and TNF-α mRNAs, in the contents of IL-1 ß and TNF-α of the EA-LI 4-PC 6 group, and in the IL-6 content of the EA-LI 18 group (P>0.05). CONCLUSIONS: EA stimulation of LI 18 and LI 4-PC 6 can significantly suppress pain reaction of neck incision in the rat, which is closely associated with its effects in down-regulating the activity of SGCs, decreasing the release of pro-inflammatory cytokines and in weakening the expression of Cx 43 in the cervical DRGs.