OBJECTIVES: To explore the mechanism of Qingda Granules (QDG) for alleviating brain damage in spontaneously hypertensive rats (SHRs). METHODS: Twelve 5-week-old SHRs were randomized into SHR control group and SHR+QDG group treated with QDG by gavage at the daily dose of 0.9 g/kg for 12 weeks. The control rats, along with 6 age-matched WKY rats, were treated with saline only. Blood pressure changes of the rats were monitored, and pathologies and neuronal apoptosis in the cerebral cortex were examined with HE staining and TUNEL staining. Cerebral cortical expressions of miR-124 and STAT3 mRNA were detected using RT-qPCR, and the protein expressions of NeuN, STAT3, Bcl-2, Bax, and cleaved caspase-3 were detected with immunohistochemistry and Western blotting. In a HT22 cell model of oxygen and glucose deprivation/reoxygenation (OGD/R), the effects of QDG on cell viability and apoptosis, expressions of miR-124 and STAT3 mRNA, and protein expressions of STAT3, Bcl-2, Bax, and cleaved caspase-3 were evaluated using CCK8 assay, Hoechst 33342 staining, RT-qPCR, and Western blotting. RESULTS: Compared with WKY rats, SHRs had significantly elevated systolic blood pressure, diastolic blood pressure and mean arterial pressure with significantly increased neuronal apoptosis in the cerebral cortex, reduced expressions of NeuN, miR-124 and Bcl-2, and enhanced expressions of STAT3, Bax and cleaved caspase-3 (P<0.05). All these changes in the SHRs were significantly ameliorated by treatment with QDG (P<0.05). In the HT22 cell model, QDG treatment obviously reduced OGD/R-induced cell apoptosis, increased the expressions of miR-124 and Bcl-2, and suppressed the elevation of protein expressions of STAT3, Bax and cleaved caspase-3. CONCLUSIONS QDG inhibits cerebral cortical neuronal apoptosis and thereby attenuates brain damage in SHR rats by modulating the miR-124/STAT3 signaling axis.
Animals ; Rats, Inbred SHR ; MicroRNAs/metabolism* ; STAT3 Transcription Factor/metabolism* ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Rats ; Apoptosis/drug effects* ; Rats, Inbred WKY ; Male ; Hypertension

Objective To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer.Methods The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2.The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments.Lactate dehydrogenase(LDH)release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells,and the secretion levels of IFN-γ,IL-21 and CCL19 were determined with enzyme-linked immunospot assay(ELISPOT)and ELISA.A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site,and the fluorescence signals were captured using a fluorescent microscopy.Results The NKP30 ligand B7H6,which was almost undetectable in normal tissues and blood cells,was highly expressed(over 90%)in lung cancer cells.Compared with NKP30 CAR-T cells and conventional T cells,IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells.The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ,IL-21 and CCL19.In the zebrafish models,CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells,but these differences were not statistically significant between the two CAR-T cells.Conclusion Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

Objective To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer.Methods The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2.The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments.Lactate dehydrogenase(LDH)release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells,and the secretion levels of IFN-γ,IL-21 and CCL19 were determined with enzyme-linked immunospot assay(ELISPOT)and ELISA.A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site,and the fluorescence signals were captured using a fluorescent microscopy.Results The NKP30 ligand B7H6,which was almost undetectable in normal tissues and blood cells,was highly expressed(over 90%)in lung cancer cells.Compared with NKP30 CAR-T cells and conventional T cells,IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells.The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ,IL-21 and CCL19.In the zebrafish models,CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells,but these differences were not statistically significant between the two CAR-T cells.Conclusion Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and is characterized by primary left ventricular hypertrophy usually caused by mutations in sarcomere genes. The mechanism underlying cardiac remodeling in HCM remains incompletely understood. An investigation of HCM through integrative analysis at multi-omics levels will be helpful for treating HCM. DNA methylation and chromatin accessibility, as well as gene expression, were assessed by nucleosome occupancy and methylome sequencing (NOMe-seq) and RNA-seq, respectively, using the cardiac tissues of HCM patients. Compared with those of the controls, the transcriptome, DNA methylome, and chromatin accessibility of the HCM myocardium showed multifaceted differences. At the transcriptome level, HCM hearts returned to the fetal gene program through decreased sarcomeric and metabolic gene expression and increased extracellular matrix gene expression. In the DNA methylome, hypermethylated and hypomethylated differentially methylated regions were identified in HCM. At the chromatin accessibility level, HCM hearts showed changes in different genome elements. Several transcription factors, including SP1 and EGR1, exhibited a fetal-like pattern of binding motifs in nucleosome-depleted regions in HCM. In particular, the inhibition of SP1 or EGR1 in an HCM mouse model harboring sarcomere mutations markedly alleviated the HCM phenotype of the mutant mice and reversed fetal gene reprogramming. Overall, this study not only provides a high-precision multi-omics map of HCM heart tissue but also sheds light on the therapeutic strategy by intervening in the fetal gene reprogramming in HCM.
Cardiomyopathy, Hypertrophic/metabolism* ; Humans ; Animals ; DNA Methylation ; Mice ; Transcriptome ; Chromatin/genetics* ; Early Growth Response Protein 1/metabolism* ; Male ; Epigenome ; Nucleosomes/genetics* ; Female ; Middle Aged ; Disease Models, Animal ; Adult

Objective: To examine the dose-response association between cardiorespiratory fitness ( CRF ) and the risk of cardiovascular and cerebrovascular diseases. Methods: A joint search was performed in Chinese and English electronic databases, including China National Knowledge Infrastructure ( CNKI ) , Wanfang Data, VIP, CBM, PubMed, Embase and Web of Science, to retrieve publications reporting the correlation between CRF and the risk of cardiovascular and cerebrovascular diseases until May, 2021. The pooled risk was estimated using the random effects model, and the dose-response association was evaluated using restricted cubic splines. The source of heterogeneity was assessed by subgroup analysis, and the stability of the results was tested by the trim-and-fill method, while the publication bias was assessed using funnel plots. Results: Totally 37 280 literatures were identified, and 23 eligible studies were finally included in the analysis, which covered 2 605 622 subjects. There were 22 publications identified as high-quality. Meta-analysis revealed that the pooled risk of cardiovascular and cerebrovascular diseases reduced by 42% in the highest CRF group relative to the lowest CRF group ( OR=0.58, 95%CI: 0.52-0.65 ), and a one metabolic equivalent ( MET ) increase in CRF caused a 10% reduction in the pooled risk of cardiovascular and cerebrovascular diseases ( OR=0.90, 95%CI: 0.88-0.92 ). There was a negative linear correlation between CRF and the incidence of cardiovascular and cerebrovascular diseases ( P=0.396 ). Subgroup analysis identified gender, sample size and study regions as possible sources of heterogeneity, and sensitivity analysis showed that the study results were stable. Conclusions There is a negative linear correlation between CRF and the risk of cardiovascular and cerebrovascular diseases, and an increase in CRF may reduce the risk of cardiovascular and cerebrovascular diseases.

Objective:To compare the clinical effects between Dynesys dynamic stabilization with microendoscopic discectomy (MED) and posterior lumbar interbody fusion (PLIF) treatment for the degenerative lumbar spinal stenosis in the elder, and to evaluate clinical advantages of Dynesys with MED according to enhanced recovery after surgery (ERAS).Methods:All of 79 cases (male 31, female 48) who suffered from the degenerative lumbar spinal stenosis were include mean age 67.32±5.88 years (from 60 to 81 years) during January 2012 to December 2017. Thirty-nine patients received Dynesys dynamic stabilization with MED (Dynesys group) and 40 cases undergone PLIF (fusion group). The operative time, blood loss, drainage, length of hospital stay, and complications were compared between the two groups. The visual analog scale (VAS), Oswestry disability index (ODI) were used to assess efficacy. The range of motion (ROM) of the lumbar spine and the adjacent segment by dynamic radiographs between two groups were reviewed to evaluate radiological results.Results:The fusion group was larger than Dynesys group in operation time (271.00±57.19 min vs. 193.85±32.17 min), blood loss (458.25±136.85 ml vs. 316.41±87.64 ml), drainage (143.12±47.46 ml vs. 101.67±31.23 ml) and length of hospital stay (15.70±3.01 d vs. 13.38±2.72 d) with statistics differences ( P<0.05). There was a significant difference in VAS and ODI between the two groups at the final follow-up, and Dynesys group was superior to the fusion group (VAS: 1.51±1.21 vs. 2.40±1.48, t=2.910, P=0.005; ODI: 11.90%±6.15% vs. 17.73%±6.85%, t=3.974, P<0.001). The lumbar ROM of Dynesys group increased comparing with the fusion group at post-operation one year (19.21°±6.08° vs. 14.08°±5.80°, t=0.425, P<0.001) and final follow-up (20.56°±6.37° vs. 16.33°±6.94°, t=2.828, P=0.006). ROM of the adjacent segment increased in fusion group at final follow-up (7.45°±2.45°) compared to pre-operation (4.68°±1.98°) and post-operation one year (4.83±1.43°) with significant difference ( F=24.437, P<0.001). The rate of epidural damage, delayed union, surface infection, internal fixation loose, thrombus, pulmonary infection, bedsore, reoperation of the fusion group were all higher than Dynesys group. Conclusion:The Dynesys with MED demonstrated better clinical and radiological results than fusion for degenerative lumbar spinal stenosis. As a method of safety and efficiency, the Dynesys and MED can speed up postoperative recovery of elderly patients in line with ERAS.

Objective: To identify pathogenic variants in 5 sporadic patients and two Chinese pedigrees affected with 17-hydroxylase deficiency (17-OHD). Methods: Peripheral blood samples were collected with informed consent. Variants of CYP17A1 gene were screened by PCR and Sanger sequencing. Suspected mutations were validated in other members of the pedigrees. Results: Gene sequencing has identified a homozygous c. 985_987delTACinsAA (Y329Kfs) mutation in exon 6 of the CYP17A1 gene in 4 patients and the sister of case 3. Case 1 was found to harbor compound heterozygous mutations c. 1459_1467del9 (p.D487_F489del) and c. 1244-3C>A. The parents and brother of cases 2 and 5 were heterozygous carriers of a c. 985_987delTACinsAA(Y329Kfs) mutation. Conclusion Mutations of the CYP17A1 gene probably underlie the pathogenesis of 17-OHD, for which c. 985_987delTACinsAA(Y329Kfs) is the most common. The c. 1244-3C>A is a novel mutation. Above results have facilitated genetic counseling for the affected families.

OBJECTIVE: To explore the genotype-phenotype correlation among 18 patients with 21-hydroxylase deficiency (21-OHD). METHODS: PCR-Sanger sequencing was used to analyze the 10 exons and flanking regions of the CYP21A2 gene among the 18 patients and 20 healthy controls. RESULTS: Seventeen patients had variants of the CYP21A2 gene. Eight patients (44.4%, 8/18) carried homozygous variants including p.Ile 173Asn (62.5%, 5/8), p.Pro31Leu (25.0%, 2/8), and IVS2-13A/C>G (12.5%, 1/8), respectively. Six patients (33.3%, 6/18) carried compound heterozygous variant, among which IVS2-13 A>G+p.Ile 173Asn were most common (50.0%). 94.4% (34/36) of the variant were pathogenic, with the most common variants being p.Ile173Asn (41.7%), IVS2-13A/C>G (19.4%), and p.Ile173Asn (7.5%). No variant was identified among the 20 healthy controls. CONCLUSION The majority of 21-OHD patients carried CYP21A2 gene variants in homozygous or compound heterozygous forms, among which the p.Ile173Asn was the most common one. There is a strong correlation between the genotypes and clinical phenotypes.
Adrenal Hyperplasia, Congenital ; genetics ; Genotype ; Humans ; Mutation ; Phenotype ; Steroid 21-Hydroxylase ; genetics

OBJECTIVE: To identify pathogenic variants in 5 sporadic patients and two Chinese pedigrees affected with 17-hydroxylase deficiency (17-OHD). METHODS: Peripheral blood samples were collected with informed consent. Variants of CYP17A1 gene were screened by PCR and Sanger sequencing. Suspected mutations were validated in other members of the pedigrees. RESULTS: Gene sequencing has identified a homozygous c.985_987delTACinsAA (Y329Kfs) mutation in exon 6 of the CYP17A1 gene in 4 patients and the sister of case 3. Case 1 was found to harbor compound heterozygous mutations c.1459_1467del9 (p.D487_F489del) and c.1244-3C>A. The parents and brother of cases 2 and 5 were heterozygous carriers of a c.985_987delTACinsAA(Y329Kfs) mutation. CONCLUSION Mutations of the CYP17A1 gene probably underlie the pathogenesis of 17-OHD, for which c.985_987delTACinsAA(Y329Kfs) is the most common. The c.1244-3C>A is a novel mutation. Above results have facilitated genetic counseling for the affected families.
Adrenal Hyperplasia, Congenital ; genetics ; Exons ; Female ; Humans ; Male ; Mutation ; Pedigree ; Steroid 17-alpha-Hydroxylase ; genetics

Objective To optimize the detection test of Pig-a gene mutation in peripheral blood of rats by enriching and detecting mutant erythrocytes, using immunomagnetic separation technique in combination with flow cytometry. Methods SD rats were administered with 20,40 and 80 mg/(kg·bw)doses of N-ethyl-N-nitrosourea(ENU) continually for 3 days. The peripheral blood samples of rats were collected on the 7th,14th and 28th days, respectively, after treatment. Immunomagnetic separation columns were used to enrich RETCD59- and RBCCD59- cells, and then flow cytometry was used to count the number of pre-column and post-column peripheral erythrocytes. Results Compared with the control group,the frequencies of RETCD59- and RBCCD59- were significantly increased in each ENU group(P<0.05). With immunomagnetic separation technique, the test of Pig-a gene mutation of a sample could be completed within 3 minutes,and the number of detected RETCD59- or RBCCD59-cells was up to 2×104or 9×104, respectively. Conclusions In this study,immunomagnetic separation in combination with flow cytometry is used to establish and optimize the Pig-a gene mutation test in rat peripheral blood,showing a high-throughput detection and improved accuracy and efficiency of the experiment.