Objective To investigate the role of protein kinase C (PKC) in induction of vascular endothelial growth factor (VEGF) secretion by isoflurane in primary cultured rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 6 groups ( n = 6 each): control group (group C), 3 different concentration isoflurane groups (group Ⅰ1-3 ), PKC inhibitor calphostin C group (group P), and PKC inhibitor + isoflurane group (group PI). The cells were exposed to 0.7%, 1.4% and 2.1% isoflurane for6 h in group Ⅰ1-3 respectivly. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L in group P. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L, then the cells were exposed to 1.4% isoflurane for 6 h in group PI. VEGF concentrations and expression of PKC isoforms were determined by ELISA and Western blot respectively. Results Compared with group C, the VEGF concentration was significantly increased in group Ⅰ2 and Ⅰ3, and PKCε expression was down-regulated in the cytoplasm while upregulated in the cytomembrane in group Ⅰ2 ( P ＜ 0.01 ), but no significant change was found in the parameters mentioned above in group Ⅰ2 ( P ＞ 0.05). PKCα, PKCδ and PKCζ expression was significantly higher in the cytoplasm than in the cytomembrane in group C and Ⅰ2. VEGF concentrations were gradually increased with the increase in isoflurane concentrations ( P ＜ 0.05). VEGF concentrations were significantly lower in group PI than in Ⅰ2 ( P ＜0.05) .Conclusion Isoflurane induces VEGF secretion in primary cultured rat cardiomyocytes through translocation of PKCε from the cytoplasm to the cytomembrane, suggesting that it is a mechanism of the cardioprotective effects of isoflurane.

Objective To study the protective effects on ovarian function by caloric restriction (CR) and its mechanism.Methods Thirty female C57BL/6 mice of 8 weeks old were randomly divided into two groups,including ad libitum (AL) group and caloric restriction (CR) group.The general situation and ovarian function of those mice were compared and evaluated.Ovarian follicles were counted by hematoxylin-eosin staining.Anti-Miillerian Hormone(AMH) mRNA expression of the ovary were detected by using real-time PCR.The concentrations of serum estradiol,progesterone of the mice were measured by ELISA.And the fertility of mice by mating trials were evaluated,SIRT3,Hypoxia inducible factor 1α (HIF-1α) and catalase (CAT) mRNA expression of the mice ovaries were detected by Real-Time PCR.Results The total follicles were 546 in CR mice and 286 in AL mice.The proportion of primordial follicles were 38.6％ (211/546)in ovaries of CR mice and 29.4％ (84/286)in ovaries of AL mice,which reached statistical difference.The proportion of atretic follicles 5.3％ (29/546) in ovaries of CR mice,compared with 16.8％ (48/286) in AL mice,was significantly decreased (P ＜ 0.05).The AMH mRNA expression in CR mice ovaries was 3.37 times of that of AL mice (P ＜ 0.05).The serum concentration of estradiol in CR mice was up to (5.3 ± 1.6) pmol/L,which was much higher than (3.6 ± 1.6) pmol/L in AL mice.While,the progesterone concentration of (0.4 ±0.3) nmol/L in CR mice was lower than (1.4 ± 0.8) nmol/L in AL mice (P ＜ 0.05).Fertility and survival of offsprings were both improved in CR mice.The expression level of SIRT3 mRNA in CR mice ovary was 1.39 times,CAT was 1.55 times and HIF-1 α was 0.31 times of those in AL mice (P ＜ 0.05).Conclusions Caloric restriction can delay the ovary aging process through reduce follicle depletion by suppressing follicle recruitment and ovulation.The function of ovarian reserve and reproductive endocrine was effectively protected.Caloric restriction can reduce the incidence of follicular atresia,its mechanism might be associated with anti-oxidative stress.

OBJECTIVETo construct adenoviral vectors expressing mature miRNA-30a and miRNA-30e.

METHODSThe target mmu-miR-30a and mmu-miR-30e genes amplified from mouse genome were digested and linked to the shuttle plasmid pSES-HUS, which was then transformed into competent AdEaseier cells for recombination. The confirmed recombinant plasmids were transfected into Hek-293 cells for production of the adenoviruses pAd-mmu-miR-30a and pAd-mmu-miR-30e. The obtained adenoviruses were used to infect Mefs cells, and the cellular expressions of mmu-miR-30a and mmu-miR-30e were detected using fluorescence quantitative PCR.

RESULTSmmu-miR-30a (357 bp) and mmu-miR-30e (324 bp) containing the restriction sites were amplified and linked to the shuttle plasmid pSES-HUS, which was successfully recombined with AdEasy1. After packaging in Hek-293 cells, the adenoviral vectors were obtained, which caused an increase of mmu-miR-30a expression by 26.46∓7.46 folds and mmu-miR-30e expression by 2.76∓0.25 folds in transfected Mefs cells.

CONCLUSIONWe have successfully constructed the adenoviral vectors expressing the mature miRNAs.

Adenoviridae ; genetics ; Animals ; Base Sequence ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; MicroRNAs ; genetics ; Plasmids

Objective: To investigate the early clinical efficacy of bone cement modified with mineralized collagen in the treatment of osteoporotic vertebral compression fractures with percutaneous vertebroplasty(PVP). Methods: All 98 cases of sin-gle vertebral osteoporotic compression fracture from June 2017 to August 2018 were studied. Forty-eight cases were treated with bone cement modified with mineralized collagen (modified group) and 50 cases were treated with traditional bone cement (traditional group). The basic clinical information including age, sex and bone mineral density of all patients were analyzed. The injectable time, volume, distribution (bone cement in the vertebra showing a whole mass without interruption or loss is known as type O while bone cement in the vertebra showing two masses with a small amount or none in the middle is known as type H) and leakage of bone cement during operation, visual analogue score(VAS), Oswestry disability index (ODI), height of anterior, middle and posterior columns of injured vertebrae and the incidence of adjacent vertebral fractures were compared between the two groups. Results: There were no significant differences in age, sex, bone mineral density T value and bone ce-ment injection volume between the two groups. VSA score, ODI, anterior and middle column heights were significantly improved on the first day and 6 months after operation (P<0.05), but there was no significant difference between the two groups (P>0.05). In-traoperative cement injectable time was shorter in the traditional group than the modified group,and there was significant different between the two groups (t=3.428, P=0.002). The incidence of adjacent vertebral re-fracture was 12% in the traditional group and 2% in the modified group. There was significant different between the two groups (χ²=7.061, P=0.029). The leakage rate of bone cement was 10% in the traditional group and 6% in the modified group, andthere was significant difference between the two groups (χ²=7.963, P=0.019). The distribution of bone cement (O/H) in the traditional group was 20/30 and that in the modified group was 19/29, and there was significant difference between the two groups (χ²=38.992, P<0.001). Conclusion Modified bone cement has the same clinical effect as traditional bone cement in relieving pain and restoring the height of injured vertebra in the treat-ment of osteoporotic vertebral compression fractures with PVP. However, the injectable time of modified bone cement is longer. The leakage rate and the incidence of re-fracture of adjacent vertebrae are significantly reduced.

Ras-associated domain family 1A (RASSF1A) genes are members of the RASSF family, which bind to Ras in a guanosine triphosphate(GTP)-dependent manner and then induce Ras-mediated apoptosis. The protein encoded by the RASSF1A gene is similar to the Ras effector protein, which can interact with DNA repair protein XPA, and can also inhibit the accumulation of cyclin D1, thereby inducing cell cycle arrest. The deletion or abnormal expression of RASSF1A gene is related to the pathogenesis of various malignant tumors, indicating that it has tumor suppressor function. RASSF1A gene methylation has been found in at least 37 tumors, and RASSF1A gene may be the most frequently described methylated gene in human cancers. In this paper, the abnormal methylation of RASSF1A gene in different malignant tumors was introduced, and the research progress of its related effects and mechanisms in malignant tumors of the respiratory system, digestive system, genitourinary system, and nervous system in recent years was reviewed, with a view to malignant tumors early diagnosis, individual molecular targeted therapy and prognostic evaluation provide important guidance.

Objective Constructing a risk prediction model of IgA nephropathy proteinuria treated by traditional Chinese medicine based on random survival forest model,Screening prognostic risk factors of IgA nephropathy proteinuria.Methods Collecting retrospectively clinical data of 129 cases diagnosed with IgA nephropathy,randomly divided them into training set(60%)and test set(40%).The risk prediction model of IgA nephropathy proteinuria was constructed in the training set with the random survival forest model,and the prognostic risk factors were screened by VIMP method.The accuracy of risk prediction model was validated in the test set with time-dependent ROC curve(tdROC).Results According to the result of VIMP,the prognostic risk factors for IgA nephropathy proteinuria are in the order of eGFR,hypertension,traditional Chinese medicine,24 hUPRO>1 g,genomo sclerosis ratio,Lee grading,fat,hyperlipidemia,hypertrophymia,hyparmane ledmia,Anemia,age and gender.The eGFR was negatively and non-linearly associated with the risk rate of developing persistent proteinuria.Glomerulosclerosis ratio greater than 0.3 is approximately linearly and positively associated with the risk rate of persistent proteinuria.Conclusion Random survival forest model has good predictive performance in the risk prediction model of IgA nephropathy proteinuria treated by traditional Chinese medicine.This risk model can determine the result of IgA nephropathy treated by traditional Chinese medicine,and which is helpful for clinical follow-up monitoring and formulation of individualized treatment plans.

Retrospective analysis of the clinical data of a child with type 102 mental retardation caused by DDX3X gene mutation in the pediatric diagnosis of the First Affiliated Hospital of Zhengzhou University in April 2019.A 2 years and 3 months old girl with " delay for more than 1 year" , using second-generation sequencing technology for full exon detection, and the result is DDX3X gene 13 th exon c. 1463G>A hybridization mutation, this is a new mutation.There are no Chinese cases reported with DDX3X gene mutations, and there are 8 related cases were reported in foreign literature, all children have different degrees of intellectual disability.So patients with unexplained intellectual disability(especially female patients) need to be wary of the possibility of DDX3X gene mutation.

ObjectiveTo explore the mechanism of Yishen Huoxue prescription in renal interstitial fibrosis （RIF） from the perspective of endothelial cell and cell energy metabolism. MethodThe model was successfully established by unilateral ureteral obstruction （UUO）. Seventy-five SPF C57BL/6 mice were randomly divided into a model group， a resveratrol group （50 mg·kg^-1·d^-1）， three Yishen Huoxue prescription low， medium， and high-dose groups （7.1， 14.2， 28.4 g·kg^-1·d^-1）， with 15 mice in each group. In addition， another 15 mice were used to prepare sham operation model. Mice in the sham operation group and the model group were gavaged with equal volume of normal saline. All mice were sacrificed on 7， 14， and 21 d after modeling. The protein expression of platelet endothelial cell adhesion molecule 31 （CD31） was detected by immunohistochemical S-P method. The expression of α-smooth muscle actin （α-SMA）， collagen Ⅳ （Col-Ⅳ）， angiopoietin 1（Ang-1） and tyrosine kinase receptors 2 （Tie-2）， vascular endothelial growth factor （VEGF）， vascular endothelial cadherin （VE-cadherin）， and occludin in renal tissues was detected by Western blotting. The mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin in renal tissues were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the levels of reactive oxygen species （ROS） in mice were detected by enzyme linked immunosorbent assay （ELISA）. ResultAs compared with the sham operation group， the expression of CD31 in renal tissues of the model group was significantly decreased and worsened with the extension of modeling time （P<0.05）， α-SAM and Col-Ⅳ protein expression levels were significantly increased （P<0.01）， but the expression of CD31 was stable in 14-21 d. ROS levels were significantly increased （P<0.01）， and the protein and mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin were significantly down-regulated （P<0.01）. As compared with the model group， the expression of CD31 was increased （P<0.05）， and α-SAM and Col-Ⅳ in the resveratrol group and the medium and high-dose Yishen Huoxue prescription groups were significantly decreased （P<0.01）. The ROS content was significantly decreased （P<0.01）， and the protein and mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin were up-regulated （P<0.01）， As compared with the resveratrol group， the protein expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin in the medium and low-dose Yishen Huoxue prescription groups were significantly different （P<0.01）. There was no significant difference in the mRNA expressions of CD31 and Ang-1/Tie-2 in the high-dose Yishen Huoxue prescription group， and no significant difference in the ROS level in the medium-dose Yishen Huoxue prescription group. ConclusionThe anti-RIF effect of Yishen Huoxue prescription may be related to promoting vascular endothelial repair， regulating mitochondrial ROS to reduce oxidative stress， protecting the integrity of renal endothelial structure， delaying cell apoptosis， and maintaining cell energy metabolism.

The thalamocortical (TC) circuit is closely associated with pain processing. The hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 channel is predominantly expressed in the ventral posterolateral thalamus (VPL) that has been shown to mediate neuropathic pain. However, the role of VPL HCN2 in modulating TC circuit activity is largely unknown. Here, by using optogenetics, neuronal tracing, electrophysiological recordings, and virus knockdown strategies, we showed that the activation of VPL TC neurons potentiates excitatory synaptic transmission to the hindlimb region of the primary somatosensory cortex (S1HL) as well as mechanical hypersensitivity following spared nerve injury (SNI)-induced neuropathic pain in mice. Either pharmacological blockade or virus knockdown of HCN2 (shRNA-Hcn2) in the VPL was sufficient to alleviate SNI-induced hyperalgesia. Moreover, shRNA-Hcn2 decreased the excitability of TC neurons and synaptic transmission of the VPL-S1HL circuit. Together, our studies provide a novel mechanism by which HCN2 enhances the excitability of the TC circuit to facilitate neuropathic pain.
Animals ; Mice ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics* ; Neuralgia ; RNA, Small Interfering ; Thalamus/metabolism* ; Up-Regulation