Objective To observe the proliferation and phenotype-switching of pulmonary arterial smooth muscle cell (PASMC) induced by hypoxia and interfered by Ad-PKGIα. And to investigate the potential regulative role of PKGIα gene in the molecule mechanism of hypoxia pulmonary vessel remodeling (HPVR). Methods To establish the pure PASMC cultured by tissue-sticking methods. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) and Western blot were used to examine the PKGIα mRNA and protein expression after PASMC were transfected by Ad-PKG. The mRNA and protein expressive change of smooth muscle α actin(SM-α-actin) determined the degree of cell phenotype-switching. The changes of PASMC proliferation were determined by flow cytometry and ~3H-TdR incorporated way. Results Ad-PKGIα could transfect into PASMC and highly express. Hypoxia down-regulated the expression of SM-α-actin protein (44. 25±5.34 in normoxia, 32. 18±4. 19 in 12 h hypoxia condition, 21.90 ±2. 44 in 24 h hypoxia condition, P < 0. 05), that could be blocked by the transfeetion of Ad-PKGIα. Hypoxia could push PASMC mitosis and proliferating(~3H-TdR incorporated way: 7570 ± 371 in normoxia,12 020± 831 in 12 h hypoxia condition,14 924 ± 1491 in 24 h hypoxia condition, P <0. 05), that could be blocked by the transfection of Ad-PKGIα, too. Conclusions The results suggested that PKGIα signaling pathway might play an important role in the molecule mechanism of HPVR. And PKGIα gene might be a target point of gene therapy.

Objective To explore the associations between coagulation factor Ⅶ (FⅦ)polymorphisms and its haplotype with risk of ischemic cerebrovascular diseases (ICVD) in Henan Han population. Methods Five hundred and twelve cases with ICVD as patient group and 560 healthy subjects as control were recruited in the study. The polymorphisms of R353Q, 5'F7 and IVS7 were detected by PCR-RFLP. The genotype frequency and allele gene frequency were compared between ICVD group and control group. The haplotype was analyzed by SHEsis software. Results The RQ genotype frequencies and Q allele frequencies of ICVD group were significantly lower than those of control group. The distribution of H7 allele frequencies and H6H7 genotype frequencies of FⅦ/IVS7 polymorphisms had significant difference between ICVD group and control group. Finally, the prevalence of R-P0-H6 haplotype in ICVD group(53. 3% )was higher than that in control group (47.5%, OR = 1. 219, 95% CI 1. 028-1. 446,P =0.023). Conclusions In Henan Han population, the Q allele of F Ⅶ/R353Q polymorphisms and the H7 allele of F Ⅶ/IVS7 polymorphisms may be protective genetic factors against ischemic cerebrovascular disease, and the R-P0-H6 haplotype may be a risk factor of ischemic cerebrovascular disease.

Objective:To analyze the activation of primary somatosensory cortex(S1)neurons in post-traumatic stress disorder(PTSD)mice after tactile stimulation of whiskers and the changes of S1 cortical neurons in PTSD mice.Methods:Using the neuron cytoskeleton-associated protein(Arc)labeling strategy and immunofluorescence staining technique,the Arc of S1 cortical neurons in PTSD mice and control mice after whisker stimulation was marked and ob-served.By analyzing the difference in the spatial expression position of Arc labeled neurons and the number of positive cells,the activation level of S1 cortical neurons in the two groups was compared and analyzed.The pyramidal neurons of S1 cortex were labeled by sparse virus labeling method,and the number of dendrites and the morphology and number distribution of dendritic spines were compared between the two groups.Results:After whisker stimulation,it was found that Arc positive neurons were distributed from shallow layer to deep layer of S1,and more densely distributed in layersⅡ/Ⅲ and V.Compared with the control group,the number of positive neurons in different layers of the PTSD group was significantly increased.The results of cell morphology and structure analysis showed that,compared with the control group,the density of dendritic spines in layer Ⅱ/Ⅲ of mice with PTSD increased,and the number of mushroom dendrit-ic spines increased,while the number of filamentous pseudopod dendritic spines decreased.The number of dendritic spines of Si V layer mushroom type and slender type was higher,but the total number of dendritic spines was not sig-nificantly different.Conclusion:After whisker stimulation in PTSD mice,S1 neurons were over-activated,and the structure and morphology of neurons changed significantly.

Objective: To investigate the mechanism of Yishen Tongluo Formula in ameliorating renal pyroptosis in diabetic nephropathy mice by regulating the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. Methods: Sixty C57BL/6 male mice were randomly divided into control (10 mice) and intervention groups (50 mice) using random number table method. The diabetes nephropathy model was established by intraperitoneally injecting streptozotocin(50 mg/kg). After modeling, the intervention group was further divided into model, semaglutide (40 μg/kg), and high-, medium-, and low-dose Yishen Tongluo Formula groups (15.6, 7.8, and 3.9 g/kg, respectively) using random number table method. The high-, medium-, and low-dose Yishen Tongluo Formula groups were administered corresponding doses of medication by gavage, the semaglutide group received a subcutaneous injection of semaglutide injection, and the control group and model groups were administered distilled water by gavage for 12 consecutive weeks. Random blood glucose levels of mice in each group were monitored, and the 24-h urinary protein content was measured using biochemical method every 4 weeks; after treatment, the serum creatinine and urea nitrogen levels were measured using biochemical method. The weight of the kidneys was measured, and the renal index was calculated. Hematoxylin and eosin, periodic acid-Schiff, periodic Schiff-methenamine, and Masson staining were used to observe the pathological changes in renal tissue. An enzyme-linked immunosorbent assay was used to detect urinary β2-microglobulin (β2-MG), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) levels. Western blotting and real-time fluorescence PCR were used to detect the relative protein and mRNA expression levels of nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3), Caspase-1, gasdermin D (GSDMD), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in renal tissue. Immunohistochemistry was used to detect the proportion of protein staining area of the TLR4, MyD88, and NF-κB in renal tissue. Results: Compared with the control group, the random blood glucose, 24-h urinary protein, serum creatinine, urea nitrogen, and renal index of the model group increased, and the urine β2-MG, NGAL, and KIM-1 levels increased. The relative protein and mRNA expression levels of NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18 in renal tissue increased, and the proportion of TLR4, MyD88, and NF-κB protein positive staining areas increased (P<0.05). Pathological changes such as glomerular hypertrophy were observed in the renal tissue of the model group. Compared with the model group, the Yishen Tongluo Formula high-dose group showed a decrease in random blood glucose after 12 weeks of treatment (P<0.05). The Yishen Tongluo Formula high- and medium-dose groups showed a decrease in 24-h urinary protein, creatinine, urea nitrogen, and renal index, as well as decreased β2-MG, NGAL, and KIM-1 levels. NLRP3, Caspase-1, GSDMD, IL-1 β, and IL-18 relative protein and mRNA expression levels were also reduced, and the proportion of TLR4, MyD88, and NF-κB protein positive staining areas was reduced (P<0.05). Pathological damage to renal tissue was ameliorated. Conclusion Yishen Tongluo Formula may exert protective renal effects by inhibiting renal pyroptosis and alleviating tubular interstitial injury in diabetic nephropathy mice by regulating the TLR4/MyD88/NF-κB signaling pathway.

ObjectiveTo investigate the effect and mechanism of Yishen Tongluo prescription in protecting mice from oxidative stress injury in diabetic kidney disease （DKD） via the nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1）/NAD（P）H quinone oxidoreductase 1 （NQO1） signaling pathway. MethodsSpecific pathogen-free （SPF） male C57BL/6 mice were assigned into a control group （n=10） and a modeling group （n=50）. The DKD model was established by intraperitoneal injection of streptozotocin. The mice in the modeling group were randomized into a model group， a semaglutide （40 μg·kg^-1） group， and high-， medium-， and low-dose （18.2， 9.1， 4.55 g·kg^-1， respectively） Yishen Tongluo prescription groups， with 10 mice in each group. The treatment lasted for 12 weeks. Blood glucose and 24-h urine protein levels were measured， and the kidney index （KI） was calculated. Serum levels of creatinine （SCr）， blood urea nitrogen （BUN）， alanine aminotransferase （ALT）， and aspartate aminotransferase （AST） were assessed. The pathological changes in the renal tissue were evaluated by hematoxylin-eosin， periodic acid-Schiff， periodic acid-silver methenamine， and Masson’s trichrome staining. Enzyme-linked immunosorbent assay kits were used to measure the levels of β2-microglobulin （β2-MG）， neutrophil gelatinase-associated lipocalin （NGAL）， kidney injury molecule-1 （KIM-1）， liver fatty acid-binding protein （L-FABP）， nitric oxide synthase （NOS）， glutathione （GSH）， total antioxidant capacity （T-AOC）， and 8-hydroxy-2'-deoxyguanosine （8-OHdG）. Immunohistochemical staining was performed to examine the expression of Kelch-like ECH-associated protein 1 （Keap1） and malondialdehyde （MDA）. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR） and Western blot were employed to determine the mRNA and protein levels， respectively， of factors in the Nrf2/HO-1/NQO1 signaling pathway. ResultsCompared with the control group， the DKD model group showed rises in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with glomerular hypertrophy， renal tubular dilation， thickened basement membrane， mesangial expansion， and collagen deposition. Additionally， the model group showed elevated levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， lowered levels of GSH and T-AOC， up-regulated expression of MDA and Keap1， and down-regulated expression of Nrf2， HO-1， NQO1， and glutamate-cysteine ligase catalytic subunit （GCLC） （P<0.05）. Compared with the model group， the semaglutide group and the medium- and high-dose Yishen Tongluo prescription groups showed reductions in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with alleviated pathological injuries in the renal tissue. In addition， the three groups showed lowered levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， elevated levels of GSH and T-AOC， down-regulated expression of MDA and Keap1， and up-regulated expression of Nrf2， HO-1， NQO1， and GCLC （P<0.05）. ConclusionYishen Tongluo prescription exerts renoprotective effects in the mouse model of DKD by modulating the Nrf2/HO-1/NQO1 signaling pathway， mitigating oxidative stress， and reducing renal tubular injuries.