Objective:To evaluate the role of nemo-like kinase (NLK) in lipopolysaccharide (LPS)-induced inflammatory responses of mouse bone marrow-derived macrophages (BMDMs) and the relationship with glycolysis.Methods:BMDMs were extracted from the wild type (WT) and NLK knockout (NLK -/-) C57BL/6 mice of either sex, aged 6-8 weeks, weighing 20-25 g, were used in this study. After induction, differentiation, maturation and purity identification, BMDMs were divided into phosphate buffer solution (PBS) control group (WT+ PBS group, NLK -/-+ PBS group, n=18) and LPS group (WT+ LPS group, NLK -/-+ LPS group, n=18) by a random number table method. LPS at a final concentration of 1 μg/ml was added to stimulate mature BMDMs for 6 h in LPS group, while the equal volume of PBS was given instead in PBS group. The cell viability was measured by Calcein/PI staining, and the expression of cellular NLK, inflammatory factors (tumor necrosis factor-alpha [TNF-α], interleukin-1 beta [IL-1β] and IL-6) and key glycolytic enzymes (hexokinase 2 [HK2], pyruvate kinase M2 (PKM2) and lactate dehydrogenase A [LDHA])mRNA was detected by real-time fluorescent quantitative polymerase chain reaction. Western blot was used to detect the expression of NLK, HK2, PKM2 and LDHA. The concentrations of TNF-α, IL-1β and IL-6 in the supernatant were determined by enzyme-linked immunosorbent assay. Glucose uptake and lactic acid production were measured by hexokinase method and colorimetric method respectively. Results:Compared with WT-type BMDMs, the expression of NLK protein and mRNA in NLK -/--type BMDMs was significantly down-regulated ( P<0.05). Compared with WT+ PBS group, the BMDM viability was significantly decreased, the expression of TNF-α, IL-1β and IL-6 was up-regulated, the concentrations of TNF-α, IL-1β and IL-6 in the supernatant were increased, the expression of NLK, HK2, PKM2 and LDHA protein and mRNA was up-regulated, and the glucose uptake and lactic acid production were increased in WT+ LPS group ( P<0.05). Compared with WT+ LPS group, the BMDM viability was significantly increased, the expression of TNF-α, IL-1β and IL-6 was down-regulated, the concentrations of TNF-α, IL-1β and IL-6 in the supernatant were decreased, the expression of NLK, HK2, PKM2 and LDHA protein and mRNA was down-regulated, and the glucose uptake and lactic acid production were decreased in NLK -/-+ LPS group ( P<0.05). Conclusions:NLK may enhance LPS-induced inflammatory responses of BMDMs by promoting glycolysis in BMDMs of mice.

ObjectiveTo observe the effect of Huayu Mingmu prescription on retinal angiogenesis and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR）-hypoxia inducible factor-1α/vascular endothelial growth factor A （HIF-1α/VEGFA） signaling pathway in diabetic retinopathy （DR） rats. MethodsSixty-four SPF-grade male SD rats were used in the study. Eleven rats were randomly selected as the normal group， while the remaining 53 rats were fed a high-sugar， high-fat diet combined with low-dose streptozotocin （STZ） intraperitoneal injection to establish a type 2 diabetes mellitus （T2DM） rat model. DR model evaluation was performed after 12 weeks of diabetes. The rats were then divided into model， low-dose， medium-dose， and high-dose groups of Huayu Mingmu prescription （9.29， 18.57， 37.14 g·kg^-1）， and a calcium dobesilate group （0.16 g·kg^-1）， with 10 rats in each group. The rats were orally administered the corresponding doses of Huayu Mingmu prescription and calcium dobesilate. The normal and model groups received equal volumes of physiological saline via gavage for 8 consecutive weeks. Retinal vascular changes were observed through fundus photography， and pathological changes in retinal tissue were evaluated using hematoxylin-eosin （HE） staining. Retinal microvascular pathological changes were examined through retinal vascular network preparation and periodic acid-Schiff （PAS） staining. Immunofluorescence （IF） was used to detect the expression of VEGFA and angiopoietin-2 （Ang-2） in retinal tissue. Western blot was employed to detect the protein expression of PI3K， Akt， mTOR， HIF-1α， VEGFA， and VEGFR2 in retinal tissue. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to assess the mRNA expression of PI3K， Akt， mTOR， HIF-1α， VEGFA， and VEGFR2 in retinal tissue. ResultsCompared with the normal group， the model group exhibited significant pathological changes in retinal tissue， including the appearance of acellular capillaries， as well as significant endothelial cell （E） proliferation and pericyte （P） loss （P<0.01）. The E/P was significantly elevated （P<0.01）. Protein and mRNA expression levels of PI3K， Akt， mTOR， HIF-1α， VEGFA， and VEGFR2 in retinal tissue were significantly increased （P<0.01）， and the expression of Ang-2 protein was significantly elevated （P<0.01）. In contrast， retinal tissue in the treatment groups showed alleviated pathological changes， with reduced endothelial cell proliferation and pericyte loss （P<0.05， P<0.01）. Among the treatment groups， the high-dose Huayu Mingmu prescription and the calcium dobesilate group exhibited a decreased E/P （P<0.01）. Protein and mRNA expression levels of PI3K， Akt， mTOR， HIF-1α， VEGFA， and VEGFR2 in retinal tissue were significantly reduced （P<0.05， P<0.01）， and the expression of Ang-2 protein was significantly decreased （P<0.01）. ConclusionHuayu Mingmu prescription can intervene in retinal neovascularization in DR rats， delay the progression of DR， and its mechanism may be related to antagonizing the PI3K/Akt/mTOR-HIF-1α/VEGFA signaling pathway.

Objective:To evaluate the role of nemo-like kinase (NLK) in lipopolysaccharide (LPS)-induced inflammatory responses of mouse bone marrow-derived macrophages (BMDMs) and the relationship with glycolysis.Methods:BMDMs were extracted from the wild type (WT) and NLK knockout (NLK -/-) C57BL/6 mice of either sex, aged 6-8 weeks, weighing 20-25 g, were used in this study. After induction, differentiation, maturation and purity identification, BMDMs were divided into phosphate buffer solution (PBS) control group (WT+ PBS group, NLK -/-+ PBS group, n=18) and LPS group (WT+ LPS group, NLK -/-+ LPS group, n=18) by a random number table method. LPS at a final concentration of 1 μg/ml was added to stimulate mature BMDMs for 6 h in LPS group, while the equal volume of PBS was given instead in PBS group. The cell viability was measured by Calcein/PI staining, and the expression of cellular NLK, inflammatory factors (tumor necrosis factor-alpha [TNF-α], interleukin-1 beta [IL-1β] and IL-6) and key glycolytic enzymes (hexokinase 2 [HK2], pyruvate kinase M2 (PKM2) and lactate dehydrogenase A [LDHA])mRNA was detected by real-time fluorescent quantitative polymerase chain reaction. Western blot was used to detect the expression of NLK, HK2, PKM2 and LDHA. The concentrations of TNF-α, IL-1β and IL-6 in the supernatant were determined by enzyme-linked immunosorbent assay. Glucose uptake and lactic acid production were measured by hexokinase method and colorimetric method respectively. Results:Compared with WT-type BMDMs, the expression of NLK protein and mRNA in NLK -/--type BMDMs was significantly down-regulated ( P<0.05). Compared with WT+ PBS group, the BMDM viability was significantly decreased, the expression of TNF-α, IL-1β and IL-6 was up-regulated, the concentrations of TNF-α, IL-1β and IL-6 in the supernatant were increased, the expression of NLK, HK2, PKM2 and LDHA protein and mRNA was up-regulated, and the glucose uptake and lactic acid production were increased in WT+ LPS group ( P<0.05). Compared with WT+ LPS group, the BMDM viability was significantly increased, the expression of TNF-α, IL-1β and IL-6 was down-regulated, the concentrations of TNF-α, IL-1β and IL-6 in the supernatant were decreased, the expression of NLK, HK2, PKM2 and LDHA protein and mRNA was down-regulated, and the glucose uptake and lactic acid production were decreased in NLK -/-+ LPS group ( P<0.05). Conclusions:NLK may enhance LPS-induced inflammatory responses of BMDMs by promoting glycolysis in BMDMs of mice.

Objective:To evaluate the role of ERBB2 interacting protein (Erbin)in liver tissues in blood coagulation of septic mice.Methods:Thirty SPF healthy male C57BL/6 mice and 30 Erbin knockout mice, aged 8-10 weeks, weighing 20-30 g, were divided into wild-type+ sham operation group (WT+ Sham group), wild-type+ sepsis group (WT+ SEP group), Erbin gene knockout+ sham operation group (EKO+ Sham group) and Erbin gene knockout+ sepsis group (EKO+ SEP group) by a random number table method, with 15 animals in each group. The mouse sepsis model was prepared by the cecal ligation and perforation method in anesthetized animals. Eye blood samples were collected at 24 h after surgery and liver tissues were obtained for microscopic examination of histopathological changes (by HE staining) which were scored and for determination of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities (by colorimetry), expression of Erbin and tissue factor (TF) (by Western blot), expression of tissue plasminogen activator (t-PA) and fibrinogen (Fib)mRNA (by quantitative polymerase chain reaction), concentrations of PT, APTT, thrombin time (TT) and Fib (by automatic coagulation analyzer), and plasma TF and interleukin-6 (IL-6) concentrations (by enzyme-linked immunosorbent assay).Results:Compared with WT+ Sham group, the lung injury score was significantly increased, the expression of TF, t-PA mRNA and FGA mRNA was up-regulated, PT, APTT and TT were prolonged, the plasma Fib concentration was increased, and the activities of ALT and AST and concentrations of TF and IL-6 in plasma were increased in WT+ SEP group ( P<0.05). Compared with WT+ SEP group, the lung injury score was significantly increased, the expression of TF, t-PA mRNA and FGA mRNA was up-regulated, PT, APTT and TT were prolonged, the plasma Fib concentration was increased, and the activities of ALT and AST and concentrations of TF and IL-6 in plasma were increased in EKO+ SEP group ( P<0.05). Conclusions:Erbin in liver tissues exerts an endogenous protective effect on blood coagulation by inhibiting the up-regulation of TF expression in septic mice.

Objective     To explore the relationship between preoperative fasting plasma glucose (FPG) and postoperative pulmonary complications (PPCs) in type 2 diabetic patients undergoing elective thoracoscopic lung resection, and provide a reference for prediction and prevention of PPCs in the clinic. Methods     A retrospective analysis was performed on the type 2 diabetic patients who underwent elective thoracoscopic lung resection for the first time in our hospital from January 2017 to March 2021. According to the level of FPG one day before the operation, the patients were divided into three groups: a hypoglycemia group (<6.1 mmol/L), a medium level blood glucose group (≥6.1 mmol/L and <8.0 mmol/L) and a high blood glucose group (≥8.0 mmol/L). Besides, the patients were divided into a PPCs group and a non-PPCs group according to whether PPCs occurred. The risk factors for PPCs were analyzed by logistic regression analysis, and the predictive value of preoperative FPG level on PPCs was estimated by the area under the receiver operating characteristic curve (AUC). Results     A total of 130 patients were included, including 75 (57.7%) males and 55 (42.3%) females with an average age of 63.5±9.0 years. Logistic regression analysis showed that compared to non-PPCs patients, the level of preoperative FPG (P=0.023) and smoking history ratio (P=0.036) were higher and the operation time was longer (P=0.004) in the PPCs patients. High FPG level on preoperative day 1 and longer operation time were associated with PPCs risk. Besides, the preoperative FPG of 6.79 mmol/L was the threshold value to predict the occurrence of PPCs [AUC=0.653, 95%CI (0.559, 0.747), P=0.003]. Conclusion     There is a certain correlation between preoperative FPG level and postoperative PPCs, which may be used as an index to predict the occurrence of PPCs.

Objective:To evaluate the relationship between Erbin and Bax/Bcl-xL-mediated cell apoptosis during sepsis-induced acute kidney injury in mice.Methods:Thirty-two SPF male wild type C57BL/6 mice, 32 SPF male Erbin (-/-) C57BL/6 mice, aged 6-8 weeks, weighing 20-30 g, were divided into 2 groups ( n=16 each) using the random number table method: wild type sham operation group (WT+ Sham group), wild type sepsis group (WT+ S group), Erbin(-/-) sham operation group (EKO+ Sham group), and Erbin(-/-) sepsis group (EKO+ S group). The sepsis model was established using the moderate cecal ligation and puncture (CLP) in anesthetized animals.The survival rates within 7 days after CLP were recorded.The serum concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), IL-1β, creatinine (Cr), blood urea nitrogen (BUN) and lactic dehydrogenase (LDH) were determined at 24 h after CLP.Then the renal tissues were taken for assessment of renal injury which was scored and for determination of the apoptosis rate (by TUNEL) and expression of cleaved-caspase-3, Bcl-xL and Bax (by Western blot). Results:Compared with sham operation groups, the survival rates were significantly decreased, the serum concentrations of IL-1β, IL-10, TNF-α, Cr, BUN and LDH, renal injury score and apoptosis rate were increased, the expression of Bax and cleaved-caspase-3 was up-regulated, and the expression of Bcl-xL was down-regulated in sepsis groups ( P<0.05). Compared with WT+ S group, the survival rates were significantly decreased, the serum concentrations of IL-1β, LDH, TNF-α, Cr and BUN and renal injury score were increased, the serum concentration of IL-10 was decreased, the apoptosis rate of renal tissues was increased, the expression of Bax and cleaved-caspase-3 was up-regulated, and the expression of Bcl-xL was down-regulated in EKO+ S group ( P<0.05). Conclusions:Erbin can inhibit Bax/Bcl-xL-mediated cell apoptosis and is involved in endogenous protective mechanism against sepsis-induced acute kidney injury in mice.

Objective:To evaluate the role of ErbB2 interacting protein (Erbin) in sepsis-associated encephalopathy (SAE) in mice and the relationship with nod-like receptor thermoprotein domain associated protein 3 (NLRP3) inflammasomes.Methods:Sixty SPF-grade healthy male wild-type C57BL/6 mice and 60 Erbin (-/-)C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups ( n=30 each) by a random number table method: wild-type sham operation group (WT+ Sham group), wild-type SAE group (WT+ SAE group), Erbin (-/-) sham operation group (EKO+ Sham group) and Erbin (-/-) plus SAE group (EKO+ SAE group). The model of SAE was established by cecal ligation and perforation in anesthetized mice.Open field test (total distance moved) was performed at 7 days after establishing the model, new object recognition test (recognition index) was performed at 8 days after establishing the model, and Morris water maze test (time of staying at target quadrant) was performed at 10 days after establishing the model.The mice were sacrificed, and hippocampal tissues were removed for microscopic examination of pathologic changes (by HE staining) and for determination of neuron count, expression of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) (by Western blot), the number of NLRP3 positive cells (by immunohistochemistry), and contents of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-18 (by enzyme-linked immunosorbent assay). The cell survival rate was calculated. Results:Compared with group WT+ Sham, the time of staying at target quadrant was significantly shortened, the recognition index and cell survival rate were decreased, the contents of IL-1β, IL-18 and TNF-α and the number of NLRP3 positive cells were increased, and the expression of NLRP3, caspase-1 and ASC was up-regulated in group WT+ SAE ( P<0.05). Compared with group EKO+ Sham, the time of staying at target quadrant was significantly shortened, the recognition index and cell survival rate were decreased, the contents of IL-1β, IL-18 and TNF-α and the number of NLRP3 positive cells were increased, and the expression of NLRP3, caspase-1 and ASC was up-regulated in group EKO+ SAE ( P<0.05). Compared with group WT+ SAE, the time of staying at target quadrant was significantly shortened, the recognition index and cell survival rate were decreased, the contents of IL-1β, IL-18 and TNF-α and the number of NLRP3 positive cells were increased, and the expression of NLRP3, caspase-1 and ASC was up-regulated in group EKO+ SAE ( P<0.05). There was no significant difference in total distance moved between the four groups ( P>0.05). Conclusion:Erbin can exert endogenous protection by inhibiting the activation of NLRP3 inflammasomes in mice with SAE.

Objective To prepare the Fe3O4-1oaded biodegradable liquid-solid phase inversion poly(lactic-co-glycolic acid) (PLGA) in situ implant for ultrasound-guided injection into nude mouse tumor model,and to investigate its clinical effect in thermomagnetic treatment of nude mice with human liver cancer SMMC-7721 cells in an alternating magnetic field.Methods An in situ implant containing 10％ Fe3O4 was prepared,and 50 μtl Fe3O4-PLGA-NMP gel was injected into the subcutaneous tissue of Kunming mice.The degradation of this material was observed for 2 consecutive months,and the changes in body weight were recorded.HE staining and Prussian blue staining were performed for the heart,liver,spleen,lung,and kidney of Kunming mice.Fresh ex vivo bovine liver was taken and cut into cubes with a dimension of 2 cmu×2 cm×2 cm and then 50 μl Fe3O4-PLGA-NMMP gel was injected;after phase inversion,the cubes ofex vivo bovine liver were heated for 1,2,3,4,and 5 minutes,respectively,and then cut open for observing the range of ablation;HE staining was also performed.Micro-CT scan was performed after ultrasound-guided injection of 50 μl Fe3O4-PLGA gel into the tumors of the nude mice,and then the nude mice were divided into treatment group and control group.The mice in the treatment group were given thermomagnetic treatment for 3 minutes,and tumor growth was observed daily.Results The biodegradation of Fe3O4-PLGA-NMMP implant showed that the subcutaneously injected material was gradually metabolized at 2 weeks after injection and that the nude mice were in good condition.The bovine liver ablation experiment showed that the range of ablation of 50 μl Fe3O4-PLGA implant reached 1.46 ± 0.11 cm.HE staining showed that part of bovine liver had coagulative necrosis.The phase inversion experiment of Fe3O4-PLGA gel showed quick liquid-solid phase inversion of the material after injection into the tumor,and the process of liquid-solid phase inversion could be monitored by ultrasound and CT.The detachment and incrustation of the tumor started at 2 days after treatment,the wound started to heal 15 days later,and the tumor tissue disappeared completely.Conclusion Ultrasound-guided injection of biodegradable Fe3O4-PLGA in situ implant combined with magnetic thermal ablation can effecfively treat human liver cancer SMMC-7721 cells in nude mice.

Humans ; Non-alcoholic Fatty Liver Disease

OBJECTIVETo explore the effect of microRNA-30a-5p (miRNA-30a-5p) on the biological behavior of human hepatoma cells.

METHODSThe liver cancer cell line SMCC-7721 cells and the normal liver cell line L02 cells (control) were transiently transfected with miRNA-30a-5p mimics and an miRNA-30a-5p inhibitor by Lipofectamine 2000 (Life Technologies). miR-30a-5p mRNA expression was detected by quantitative real-time (q)PCR. Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay and apoptosis was assessed by flow cytometry.Invasion and migration were measured by transwell chamber assays. The SMCC-7721 cells was injected subcutaneously into nude mice to establish a tumor animal model.

RESULTSThe SMCC-7721 cells transfected with miRNA-30a-5p mimics showed significantly higher miRNA-30a-5p mRNA expression than the non-transfected SMCC-7721 cells and the transfected control L02 cells (P<0.01). The miRNA-30a-5p mRNA expression was significantly lower in the SMCC-7721 cells transfected with the miRNA-30a-5p inhibitor than the non-transfected SMCC-7721 cells the control L02 cells (P<0.01). The overexpression of miRNA-30a-5p inhibited the viability, colony formation rate, and invasion and migration abilities, as shown in the cells transfected with the miRNA-30a-5p mimics (P<0.05); in addition, the miRNA-30a-5p promoted proliferation of cells (P<0.05), as shown by more S phase cells detected by flow cytometry. SMCC-7122 cells transfected with miRNA-30a-5p mimics produced tumors with significantly higher average weight than tumors produced by SMCC-7122 cells that were untransfected or transfected with empty vector (both P<0.01).

CONCLUSIONOverexpression ofmiR-30a-5p had an inhibitory effect on cell proliferation, induced apoptosis, increased the number of cells in S phase, and markedly inhibited invasion and migration of SMCC-7721 HCC cells in vitro and in vivo.

Animals ; Apoptosis ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; Mice ; Mice, Nude ; MicroRNAs ; Neoplasm Invasiveness ; Transfection