OBJECTIVE: To investigate the effects of calcitonin gene-related peptide (CGRP) on autophagy in hypoxic/reoxygenated (H/R) cardiomyocytes and its relationship with the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. METHODS: The rat cardiomyocyte cell line H9c2 was routinely cultured in vitro and passaged for experiments when the cells grew to 80% fusion. (1) CGRP dosage screening experiment: the cells were divided into blank control group, H/R group and different dosages of CGRP pretreatment groups. H9c2 cells were placed in a closed hypoxia chamber for 2 hours and then reoxygenated in a conventional incubator for 12 hours to prepare the H/R model. The CGRP pretreatment groups were pretreated with 0.01, 0.1, 0.5, 1, 5, and 10 μmol/L CGRP before the modeling process. The blank control group was not given any treatment. Cell counting kit-8 (CCK-8) was used to detect the cell survival rate, and the most suitable drug dosage was screened out. (2) Intervention experiment: H9c2 cells were divided into blank control group, H/R group, CGRP+H/R group, and CGRP+PI3K target inhibitor ly294002 (LY)+H/R group. H/R group was prepared as cellular H/R model. CGRP (1 μmol/L) alone or in combination with LY (10 μmol/L) was administered to CGRP+H/R group and CGRP+LY+H/R group, respectively, prior to the preparation of cellular H/R model. The blank control group was cultured routinely without treatment. The cell survival rate was detected by CCK-8. The level of lactate dehydrogenase (LDH) release was detected by colorimetric assay. The expressions of autophagy-related proteins [autophagy effector protein Beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II), autophagy protein p62] and PI3K/Akt/mTOR signaling pathway proteins [phosphorylated Akt (p-Akt), phosphorylated mTOR (p-mTOR)] were detected by Western blotting. RESULTS: (1) Results of CGRP dosage screening experiment: compared with the blank control group, the cell survival rate of the H/R group decreased significantly; and after giving 0.1, 0.5, 1, 5 μmol/L CGRP for pretreatment, the cell survival rate increased significantly, and intervention effect of 1 μmol/L CGRP was the best, and the difference was statistically significant when compared with that of the H/R group [(74.23±6.18)% vs. (23.43±4.09)%, P < 0.01], so it was used as the intervention dosage for the subsequent experiment. (2) Intervention experiment results: compared with the blank control group, the cell survival rate in the H/R group was significantly reduced, the level of LDH release was significantly increased, the protein expressions of Beclin-1 and LC3-II were significantly increased, and the protein expressions of p62, p-Akt and p-mTOR were significantly reduced, indicating that the death of cardiomyocytes occurred after the treatment of H/R and was accompanied by the elevation of autophagy level, and this process was associated with the activation of PI3K/Akt/mTOR signaling pathway. Compared with the H/R group, CGRP pretreatment increased cell survival rate [(76.02±2.43)% vs. (46.15±3.29)%, P < 0.01], decreased the level of LDH release (U/L: 169.83±11.65 vs. 590.17±34.50, P < 0.01), and down-regulated the protein expressions of Beclin-1 and LC3-II [Beclin-1 protein (Beclin-1/β-actin): 1.27±0.15 vs. 1.93±0.19, LC3-II protein (LC3-II/LC3-I): 1.27±0.13 vs. 1.98±0.18, both P < 0.01], up-regulated the protein expressions of p62, p-Akt, p-mTOR [p62 protein (p62/β-actin): 0.96±0.02 vs. 0.63±0.05, p-Akt protein (p-Akt/Akt): 0.76±0.04 vs. 0.48±0.02, p-mTOR protein (p-mTOR/mTOR): 1.13±0.09 vs. 0.68±0.15, all P < 0.05], suggesting that CGRP was able to reduce the H/R-induced cardiomyocyte injury, and this process was accompanied by a decrease in the level of cellular autophagy and activation of the PI3K/Akt/mTOR signaling pathway. Compared with the CGRP+H/R group, the cell survival rate was significantly lower than that in the CGRP+LY+H/R group [(56.95±6.63)% vs. (76.02±2.43)%, P < 0.01], LDH release level was significantly higher (U/L: 436.00±27.44 vs. 169.83±11.65, P < 0.01), and the protein expressions of Beclin-1 and LC3-II were significantly up-regulated [Beclin-1 protein (Beclin-1/β-actin): 1.63±0.12 vs. 1.27±0.15, LC3-II protein (LC3-II/LC3-I): 1.61±0.13 vs. 1.27±0.13, both P < 0.01], and significantly down-regulated p62, p-Akt, and p-mTOR protein expressions [p62 protein (p62/β-actin): 0.57±0.09 vs. 0.96±0.02, p-Akt protein (p-Akt/Akt): 0.45±0.01 vs. 0.76±0.04, p-mTOR protein (p-mTOR/mTOR): 0.66±0.06 vs. 1.13±0.09, all P < 0.05], suggesting that PI3K-targeted inhibitor was able to reverse the protective effect of CGRP on H/R cells. CONCLUSIONS CGRP pretreatment attenuated H/R-induced cardiomyocyte injury, increased cell survival rate, and reduced cellular LDH release. This effect may be achieved through inhibiting the activation of PI3K/Akt/mTOR signaling pathway.
Animals ; Myocytes, Cardiac/drug effects* ; Signal Transduction/drug effects* ; Rats ; TOR Serine-Threonine Kinases/metabolism* ; Calcitonin Gene-Related Peptide/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Cell Line ; Cell Hypoxia ; Phosphatidylinositol 3-Kinases/metabolism*

Objective:To analyze the genetic characteristics of the prevalent strains of Varicella-Zoster virus (VZV) in the population of Dali, Yunnan, and to understand its evolutionary status in the population of Dali.Methods:Herpes fluid and 163 sera were collected from 249 patients clinically suspected to have varicella or herpes zoster in the Department of Dermatology of the Second People′s Hospital of Dali city, Yunnan province, China, from 2023 to 2024. The levels of VZV-specific IgG and IgM antibodies in serum were detected using enzyme-linked immunoassay. Viral DNA was extracted from the herpes fluid, and the cycle threshold ( Ct) of the samples was detected using quantitative real-time polymerase chain reaction (qPCR), and some samples with Ct ≦ 22 were selected for sequencing by next-generation sequencing technology (next-generation sequencing). Next-generation sequencing (NGS) was used to obtain 90 whole genome sequences of VZV, and the sequencing result were compared with the sequences of reference strains for multiple sequence comparison and evolutionary analysis. Snapgene was used to translate the nucleotides into amino acids, and the result were compared with the amino acid sequences of the reference strain. Results:Of the 90 VZV whole-genome sequences, one whole-genome sequence was from an adult varicella patient, and the remaining 89 whole-genome sequences were from herpes zoster patients. The serum-specific IgG antibody positivity rate was 99.4%, and the IgM antibody positivity rate was 52.8%. The result of both single nucleotide polymorphism (SNPs) site typing and genome-wide phylogenetic tree analysis showed that 83 of the 90 VZV whole-genome sequences in this study were on the same branch as Clade 2, and 7 VZV whole-genome sequences were on the branch of Clade 9.Conclusions:The main endemic branch in Dali region in 2023-2024 was Clade 2, with the emergence of Clade 9 branch; there were amino acid mutations in the proteins encoded by ORF22 and ORF68 in 83 VZV whole genome sequences of Clade 2 branch, and the mutations did not cause significant changes to the protein structure.

Objective:To analyze the genetic characteristics of the prevalent strains of Varicella-Zoster virus (VZV) in the population of Dali, Yunnan, and to understand its evolutionary status in the population of Dali.Methods:Herpes fluid and 163 sera were collected from 249 patients clinically suspected to have varicella or herpes zoster in the Department of Dermatology of the Second People′s Hospital of Dali city, Yunnan province, China, from 2023 to 2024. The levels of VZV-specific IgG and IgM antibodies in serum were detected using enzyme-linked immunoassay. Viral DNA was extracted from the herpes fluid, and the cycle threshold ( Ct) of the samples was detected using quantitative real-time polymerase chain reaction (qPCR), and some samples with Ct ≦ 22 were selected for sequencing by next-generation sequencing technology (next-generation sequencing). Next-generation sequencing (NGS) was used to obtain 90 whole genome sequences of VZV, and the sequencing result were compared with the sequences of reference strains for multiple sequence comparison and evolutionary analysis. Snapgene was used to translate the nucleotides into amino acids, and the result were compared with the amino acid sequences of the reference strain. Results:Of the 90 VZV whole-genome sequences, one whole-genome sequence was from an adult varicella patient, and the remaining 89 whole-genome sequences were from herpes zoster patients. The serum-specific IgG antibody positivity rate was 99.4%, and the IgM antibody positivity rate was 52.8%. The result of both single nucleotide polymorphism (SNPs) site typing and genome-wide phylogenetic tree analysis showed that 83 of the 90 VZV whole-genome sequences in this study were on the same branch as Clade 2, and 7 VZV whole-genome sequences were on the branch of Clade 9.Conclusions:The main endemic branch in Dali region in 2023-2024 was Clade 2, with the emergence of Clade 9 branch; there were amino acid mutations in the proteins encoded by ORF22 and ORF68 in 83 VZV whole genome sequences of Clade 2 branch, and the mutations did not cause significant changes to the protein structure.

Objective To investigate the prognostic value of the age-adjusted Charlson comorbidity index(ACCI)in obstructive sleep apnea(OSA)patients over 60 years old.Methods A total of 1183 elderly OSA patients admitted in sleep centers of six hospitals between January 2015 and October 2017 were consecutively enrolled,and they were divided into low(ACCI＜5,847 cases)and high ACCI group(ACCI≥5,336 cases)based on the ACCI threshold.Demographic character-istics,clinical features,sleep parameters,and medical history were obtained from their medical re-cords.ACCI was calculated.All of them patients were followed up,and all-cause mortality was re-garded as the primary outcome.ROC curves were plotted,the AUC values were calculated,and log-rank test and Cox proportional hazards regression model were used for analysis with 5 as the threshold of ACCI.Results During a median follow-up of 43 months,63 deaths(5.3％)occurred.ROC curve analysis showed the optimal cut-off value of ACCI was 4.5,with an AUC value of 0.70(95％CI:0.63-0.77,P＜0.01).Compared with the low ACCI group,the high ACCI group showed significant increases in age,body mass index,smoking,alcohol consumption,hyperten-sion,average apnea time,creatinine,cystatin C,average platelet volume,and all-cause mortality rate,while the average SpO2 and minimum SpO2 were significantly reduced(P＜0.05,P＜0.01).Multivariate Cox regression analysis revealed that total sleep time(HR=1.258,95％CI:1.053-1.503,P=0.011),mean corpuscular volume(HR=1.047,95％CI:1.007-1.087,P=0.019),and ACCI(HR=1.585,95％CI:1.384-1.815,P=0.001)were independent predictors of all-cause mortality in elderly OSA patients.Kaplan-Meier survival analysis indicated that the 6-year surviv-al rate was 56.0％and 92.2％for the high and low ACCI groups,respectively.Stratified analysis by gender and severity suggested that the cumulative survival rate was obviously higher in the low ACCI group than the high ACCI group(Plog rank＜0.01).After adjusting for confounding varia-bles,the mortality risk of the high ACCI group was 3.32 times higher than that of the low ACCI group(95％CI:1.91-5.77,P＜0.01).Conclusion ACCI is a risk predictor for all-cause mortali-ty in elderly OSA patients,and it can provide reference for clinical decision making in elderly OSA patients.

Objective To investigate the prognostic value of the age-adjusted Charlson comorbidity index(ACCI)in obstructive sleep apnea(OSA)patients over 60 years old.Methods A total of 1183 elderly OSA patients admitted in sleep centers of six hospitals between January 2015 and October 2017 were consecutively enrolled,and they were divided into low(ACCI＜5,847 cases)and high ACCI group(ACCI≥5,336 cases)based on the ACCI threshold.Demographic character-istics,clinical features,sleep parameters,and medical history were obtained from their medical re-cords.ACCI was calculated.All of them patients were followed up,and all-cause mortality was re-garded as the primary outcome.ROC curves were plotted,the AUC values were calculated,and log-rank test and Cox proportional hazards regression model were used for analysis with 5 as the threshold of ACCI.Results During a median follow-up of 43 months,63 deaths(5.3％)occurred.ROC curve analysis showed the optimal cut-off value of ACCI was 4.5,with an AUC value of 0.70(95％CI:0.63-0.77,P＜0.01).Compared with the low ACCI group,the high ACCI group showed significant increases in age,body mass index,smoking,alcohol consumption,hyperten-sion,average apnea time,creatinine,cystatin C,average platelet volume,and all-cause mortality rate,while the average SpO2 and minimum SpO2 were significantly reduced(P＜0.05,P＜0.01).Multivariate Cox regression analysis revealed that total sleep time(HR=1.258,95％CI:1.053-1.503,P=0.011),mean corpuscular volume(HR=1.047,95％CI:1.007-1.087,P=0.019),and ACCI(HR=1.585,95％CI:1.384-1.815,P=0.001)were independent predictors of all-cause mortality in elderly OSA patients.Kaplan-Meier survival analysis indicated that the 6-year surviv-al rate was 56.0％and 92.2％for the high and low ACCI groups,respectively.Stratified analysis by gender and severity suggested that the cumulative survival rate was obviously higher in the low ACCI group than the high ACCI group(Plog rank＜0.01).After adjusting for confounding varia-bles,the mortality risk of the high ACCI group was 3.32 times higher than that of the low ACCI group(95％CI:1.91-5.77,P＜0.01).Conclusion ACCI is a risk predictor for all-cause mortali-ty in elderly OSA patients,and it can provide reference for clinical decision making in elderly OSA patients.

Objective:To study the changes in long non-coding RNA C2dat1 expression in kidney tissues of rats at different stages of diabetic kidney disease (DKD) and its relationship with renal interstitial fibrosis.Methods:Forty-eight male SD rats were randomly divided into two groups with 24 rats in each group: control group and DKD group. The rats in the control group were fed with ordinary diet, while those in the DKD group were fed with high-fat diet and drank water freely. After eight weeks of feeding, the rats were fasted for 12 h with free access to water. Then, the DKD group was given a one-time intrabitoneal injection of streptozotocin and the control group was given an equal dose of sodium citrate buffer. After 72 h, the random peripheral blood glucose concentration (≥ 16.7 mmol/L for three consecutive days) and urine sugar (positive) were tested to assess the establishment of the diabetes model. Urine, blood and kidney samples were collected at 3, 6, 9 and 12 weeks. The urinary protein excretion rate within 24 h, urinary creatinine and serum total cholesterol were measured by automatic biochemical apparatus. Pathological changes in kidney tissues were observed by HE staining. The expression of calcium/calmodulin-dependent protein kinase Ⅱ delta (CaMK2D), p65, p50, α-SMA and E-cardherin was detected by immunohistochemistry. Quantitative real-time PCR (qPCR) was used to detect the expression of lncRNA C2dat1 and CaMK2D. The relationship of lncRNA C2dat1 with α-SMA, E-cardherin and CaMK2D was analyzed by correlation analysis. In in vitro experiment, renal tubular epithelial cells HK-2 were induced by high glucose. The expression of lncRNA C2dat1 and CaMK2D in HK-2 cells was detected by qPCR after 24, 48 and 72 h of intervention. Results:The rats in the DKD group showed typical symptoms such as polydipsia, polyphagia, significant weight loss and increased blood glucose as compared with the rats in the control group. Results of the biochemical tests revealed that compared with the control group, the DKD group had increased 24 h excretion rate of urinary protein, decreased urinary creatinine and up-regulated total cholesterol. HE staining showed that the rats in the control group had intact glomeruli, normal basement membrane and no mesangial hyperplasia or inflammatory cell infiltration. However, enlarged glomeruli and evenly thickened basement membrane were observed in the DKD group. Immunohistochemistry indicated that the expression of CaMK2D, p50 and α-SMA was higher in the DKD group than in the control group, while the expression of E-cardherin was lower in the DKD group. qPCR results showed that the expression of lncRNA C2dat1 and CaMK2D at mRNA level was higher in the DKD group than in the control group. In in vitro experiment, the expression of lncRNA C2dat1 and CaMK2D at mRNA level was also higher in HK-2 cells induced by high glucose than in the control group. Correlation analysis indicated that lncRNA C2dat1 was positively correlated with α-SMA and CaMK2D, but negatively correlated with E-cardherin. Conclusions:During the progression of DKD, the high expression of lncRNA C2dat1 might promote diabetic renal interstitial fibrosis by regulating the expression of CaMK2D to activate the NF-κB signaling pathway.

Objective:To evaluate the mid- and long-term outcomes of Dynesys hybrid surgery (in some segments act as a non-fusion device, in other segments act as an alternative of rigid fixation in combination with interbody fusion) in the treatment of multi-segmental lumbar degenerative disease (LDD).Methods:The data of 27 patients who received Dynesys hybrid surgery (hybrid group) for the treatment of LDD from May 2011 to September 2016 and completed the follow-up were retrospectively analyzed. Among them, there were 8 males and 19 females; their average age was 59.1±11.9 years (23-78 years). Main diagnosis: 13 cases of lumbar spinal stenosis, 14 cases of lumbar disc herniation; 4 cases of combined lumbar dynamic position instability, 7 cases of combined lumbar spondylolisthesis. There were 15 cases of two-segment disease, 11 cases of three-segment disease, and 1 case of four-segment disease. Segments distribution: 9 cases of L 3-L 5, 6 cases of L 4-S 1, 7 cases of L 3-S 1, 4 cases of L 2-L 5, and 1 case of L 2-S 1. Midline incision was used to exposure, followed by bilateral pedicle screws implantation, and interbody fusion cage with bone grafting were performed at the fusion level. Twenty-seven patients who underwent TLIF+rigid internal fixation during the same period were included as the control group. Clinical outcomes were measured by visual analog scale (VAS) for low back pain and leg pain, and Oswestry disability index (ODI). Radiological outcomes included fusion rate, intervertebral disc height (DH) of surgical segments and the proximal adjacent segment, range of motion (ROM) of non-fusion segments and the proximal adjacent segment. At the same time, the occurrence of complications was observed. Results:Patients of Hybrid group and control group were followed up for an average of 83.8±20.9 months (48-112 months) and 87.3±16.2 months (53-114 months), respectively. Baseline data of the two groups (average follow-up time, age, gender, surgical level, diagnosis) showed no significant difference. The operation time (183.0±27.8 min) and intraoperative blood loss (301.9±178.9 ml) in the hybrid group were significantly lower than those in the control group (operation time t=2.337, P=0.023; blood loss t=2.706, P=0.01). At the final follow-up, the VAS scores of low back pain and leg pain (low back pain t=12.164, P<0.001; leg pain t=20.603, P<0.001), as well as ODI were significantly improved ( t=22.827, P<0.001). A total of 32 segments received TLIF+Dynesys stabilization and 35 segments received Dynesys non-fusion stabilization in the hybrid group, with 28 segments (87.5%) achieved solid fusion at 1-year follow-up. There were 67 fusion segments in the control group, and the fusion rate at 1-year follow-up was 85.1%. DH of non-fusion segments were lower than that before surgery with statistical significance at final follow-up ( t=2.647, P=0.012), while DH of the fusion segments in the hybrid group and the surgical segments in the control group increased compared with that before surgery at the final follow-up. A certain degree of ROM (2.4°±1.5°) was retained of the non-fusion segments at the final follow-up; the ROM of proximal adjacent segments of non-fused segments was significantly smaller than that of proximal adjacent segments of fused segments ( t=2.126, P=0.044). In the hybrid group, screw loosening occurred in 4 patients (8 screws) and adjacent segment degeneration (ASD) occurred in 5 patients. In the control group, screw loosening occurred in 3 patients (6 screws), while ASD occurred in 8 patients. No screw fracture was observed during the follow-up period and no patients received reoperation. Conclusion:Hybrid surgery of Dynesys stabilization combined with interbody fusion is a safe and effective method for the treatment of multi-segmental LDD. Compared with multi-segmental fusion, this lumbar hybrid surgery has the advantages of less trauma and retaining partial segmental ROM.

ObjectiveTo screen out the mRNAs involved in the resistance of hepatoma cells to anlotinib using ceRNA microarray. MethodsHigh-dose shock combined with low-dose induction was used to culture hepatoma cells resistant to anlotinib, and CCK8 assay was used to verify the difference in the proliferation of drug-resistant hepatoma cells treated by anlotinib. The ceRNA microarray was used to screen out the differentially expressed genes between drug-resistant hepatoma cells and normal hepatoma cells, and real-time PCR was used to verify the differentially expressed genes detected by some microarrays. the independent samples t-test was used for comparison of continuous data between two groups, and the Kaplan-Meier method was used to analyze the overall survival of hepatoma cells samples, and the log-rank test was used to compare survival rates. Fisher’s exact test was used for chip screening. ResultsThere was a significant difference in gene expression between drug-resistant hepatoma cells and normal hepatoma cells, and 10 genes with the greatest difference were screened out for analysis by reducing the range. There were 4 genes associated with drug resistance and tumor growth, i.e., BIRC2, BIRC7, ABCC2, and MAPK8. There were significant reductions in the expression levels of BIRC2, ABCC2, and MAPK8 (P=0001 4, 0001 2, and 0.011 8), and there was a significant increase in the expression of BIRC7 (P＜0.001). The results of real-time PCR were consistent with those of microarray (t=10.74,32.65,18.34, and 2.80; P=0.000 4, 0.000 1, 0.000 1, and 0.044 8). The high expression of BIRC7 and the low expression of MAPK8 were associated with the significant reduction in survival time (P=0.022 0 and 0.005 6). ConclusionBIRC2, BIRC7, ABCC2, and MAPK8 are differentially expressed between anlotinib-resistant hepatoma cells and normal hepatoma cells and may be involved in the resistance of hepatoma cells to anlotinib.

[This corrects the article DOI: 10.1016/j.apsb.2019.03.006.].

Nanomedicine is charactered with a high specific surface area, diversified structure and function, and charged surface. It can realize the targeted therap by functional modification of surface or introducing the stimuli-responsive unit. Therefore, nanomedicine is increasingly being concerned. Because nanomedicine can accumulate efficiently in the lungs, drug delivery systems based on nanotechnology have broad prospects in the field of the diagnosis, prevention, and treatment in pediatric lung diseases. Herein, we reviewed the research progress of nanomedicine in pediatric lung diseases, especially in respiratory syncytial virus infection and cystic fibrosis.