1.Spatio-temporal Expression of Heterologous Oct-4 Promoter in Preimplantation Embryos of Pig, Rabbit and Mouse
Ying ZHANG ; Fangyu SHI ; Shaorong GAO ; Aixing LIN
Progress in Biochemistry and Biophysics 2006;0(09):-
The transcription factor Oct-4 is expressed specifically in mammalian preimplantation embryos and its function is related to the maintenance of embryonic stem cell pluripotency. The functional role of the heterogenous expression of Oct-4 remains unclear however. A GFP reporter construct, pOct-4(p)-GFP was generated, containing the upstream regulatory regions of bovine Oct-4 gene and its expression pattern was evaluated in the developing embryos of mouse, pig and rabbit following intracytoplasmic sperm injection. GFP fluorescence was visible early at the 2-cell stage and then became stronger in the blastocysts of all three species. However, the distribution of the GFP signals was restricted to the cells of inner cell mass and no fluorescence was detectable in trophectoderm cells. These results suggest that the bovine Oct-4 promoter is functional and that its embryonic expression activity is similar in different mammalian species.
2.Study on Molecular Mechanism of Reactive Changes in Kidney, Skeleton and Brain of Mice Models in High Altitude Hypoxia Environment
Fangyu AN ; Yali LUO ; Yongqi LIU ; Yingdong LI ; Xuesong LIU ; Xufeng SHI ; Zhuoyue GAO
Chinese Journal of Information on Traditional Chinese Medicine 2016;(1):60-64
Objective To observe the molecular mechanism of adaptive response of the kidney and skeleton and brain issues in the high altitude hypoxia; To discuss the unity of yin and yang oscillation relationship of kidney and brain marrow.Methods SPF KM mice were randomly divided into control group and model group according to random number table method. Mice in the model group were exposed to high altitude hypoxia cabin for successive 21 d. On the 22nd day, mice got out of the cabin and their body weight was measured, and then they were put to death through eyeball blood sampling. The activities of lactic LDH and Na+-K+-ATPase in brain tissue were detected by spectrophotometric colorimetry. The PFK activities of brain and skeletal muscle were detected by ELISA. Meanwhile the contents of EPO and EPOR in the kidney were measured by ELISA. The mRNA expressions of HIF-1α and AQP-4 in brain were assessed by RT-PCR. At the same time, the protein expressions of HIF-1α and AQP-1 in brain and the protein expression of Mb in skeletal muscle were detected by Western blot.Results Compared with the normal group, the LDH and PFK in brain tissue and the content of EPO in kidney tissue were all raised in the model group(P<0.05). Meanwhile the mRNA expressions of HIF-1α and AQP-4 and the protein expressions of HIF-1α and AQP-1 in brain were all increased in the mice from the model group; the activities of PFK and the protein expression of Mb in skeletal muscle were also raised in the model group. But the activity of Na+-K+-ATPase in brain tissue and the content of EPOR in kidney tissue both decreased in the model group (P<0.05,P<0.01).Conclusion Adaptive response and the unity of yin and yang oscillation relationship between kidney, skeleton and brain tissue happen in high altitude hypoxia.
3. Inhibition of the TGF-β/Smad3 signaling pathway by SB431542: A study of the intervention effect of SB431542 on silicotic fibrosis in rats
Qianwen ZHOU ; Fangyu GAO ; Di TIAN ; Herong LIU ; Faxuan WANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2019;37(11):801-805
Objective:
To investigate the intervention effect of SB431542, which inhibits the TGF-β/Smad3 signaling pathway, on silicotic fibrosis in rats.
Methods:
A total of 40 specific pathogen-free Sprague-Dawley rats were divided into normal saline control group, model group, SB431542 inhibitor group, and SB431542 inhibitor control group using a random number table, with 10 rats in each group. All rats except those in the normal saline control group were given non-exposed single intratracheal instillation of free silicon dioxide dust suspension 1 mL (50 mg/mL) ; the rats in the SB431542 inhibitor group were given intraperitoneal injection of SB431542 (5 mg/kg) on days 7 and 30 after dust exposure, those in the SB431542 inhibitor control group were given intraperitoneal injection of SB431542 cosolvent (5 mg/kg) on days 7 and 30 after dust exposure, and those in the normal saline control group were given intratracheal instillation of an equal volume of normal saline (5 mg/kg). On day 60 after dust exposure, the paraffin-embedded section of the right upper lobe of lung was collected for HE staining; the left upper lobe of lung was collected to measure the mRNA levels of fibronectin (FN) , collagen type I (COL-I) , and collagen type III (COL-III) by quantitative real-time PCR; the right inferior lobe of lung was collected to measure the protein levels of FN, COL-I, COL-III, phosphorylated Smad3 (p-Smad3) , and Smad3.
Results:
Compared with the normal saline control group, the model group had nodules with various sizes in lung tissue, with rupture of some alveolar septa, emphysema changes, and pulmonary interstitial fibrosis, as well as significant increases in the mRNA expression of FN, COL-I, and COL-III and the protein expression of FN, COL-I, COL-III, p-Smad3, and Smad3 in lung tissue (
4.Application effect of multimodal quantitative rehabilitation exercise in patients with ankylosing spondylitis
Cuirong GAO ; Hui LI ; Fangyu LI ; Yumin SHAO
Chinese Journal of Practical Nursing 2023;39(8):593-598
Objective:To explore the application effect of multimodal quantitative rehabilitation exercise in patients with ankylosing spondylitis and to provide reference for patients′ rehabilitation exercise.Methods:The quasi-experimental study method was used to select 78 patients with ankylosing spondylitis admitted to Rheumatology and Immunology Department of First Affiliated Hospital of Xinjiang Medical University from February 2021 to February 2022 as the research objects. The 39 patients admitted from February 2021 to August 2021 as the control group, and 39 patients admitted from September 2021 to February 2022 as the experimental group. The control group adopted conventional rehabilitation exercise program, and the experimental group adopted multimodal quantitative rehabilitation exercise program. The Bath ankylosing spondylitis disease activity index, Bath ankylosing spondylitis function index, inflammatory factors after 6 months of intervention were compared between the two groups.Results:The Bath ankylosing spondylitis disease activity index in the experimental group after 6 months of intervention was (2.35 ± 0.81) points, and that in the control group was (3.47 ± 1.04) points, with a statistically significant difference ( t = 4.02, P<0.05). The Bath ankylosing spondylitis disease activity index in the two groups were analyzed by repeated measurement variance. The differences of time effect, inter group effect and interaction effect were statistically significant ( Fgroup = 11.27, Ftime = 62.05, Finteraction = 5.47, all P<0.05). The Bath ankylosing spondylitis function index in the experimental group after 6 months of intervention was (2.11 ± 0.32) points, and that in the control group was (3.07 ± 0.58) points, with a statistically significant difference ( t = 4.03, P<0.05). The Bath ankylosing spondylitis function index in the two groups were analyzed by repeated measurement variance. The differences in time effect, inter group effect and interaction effect were statistically significant ( Fgroup = 21.44, Ftime = 42.25, Finteraction = 16.67, all P<0.05). After 6 months of intervention, C-reactive protein, interleukin-6, transforming growth factor β, tumor necrosis factor-α were (43.15 ± 2.21) mg/L, (3.28 ± 0.85) mg/L, (41.67 ± 9.04) ng/L, (176.63 ± 20.15) ng/L respectively in the experimental group, and (50.12 ± 1.67) mg/L, (5.27 ± 0.68) mg/L, (48.65 ± 8.96) ng/L, (194.56 ± 19.45) ng/L respectively in the control group. There was a statistically significant difference in the content of inflammatory factors between the two groups ( t values were 2.05-4.45, all P<0.05). Conclusions:Multimodal quantitative rehabilitation exercise can improve the physiological function of ankylosing spondylitis patients′spine, reduce the disease activity of patients, and promote the rehabilitation of patients.
5.Effect of proprotein convertase subtilisin/kexin type 9 on platelet activation associated with sepsis
Yuru GAO ; Jing WANG ; Xiao HUANG ; Jinlian XU ; Jie LI ; Dakang SUN ; Dong HAO ; Fangyu NING ; Xiaozhi WANG ; Tao WANG
Chinese Critical Care Medicine 2023;35(8):807-812
Objective:To investigate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on platelet activation in sepsis.Methods:① Clinical trial: a prospective study was conducted. Patients with sepsis and septic shock aged ≥ 18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects. Healthy subjects in the same period were taken as healthy control group. Platelet count (PLT) in the first routine blood test after admission was recorded. Venous blood was taken 1 day after diagnosis, and serum PCSK9 level was determined by enzyme-linked immunosorbent assay (ELISA). The differences of PCSK9 level and PLT between the two groups were compared, and subgroup analysis was conducted based on PLT for patients with sepsis. The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method. ② Animal experiment: 80 male C57BL/6 mice were randomly divided into control group, sepsis model group [lipopolysaccharide (LPS) group], PCSK9 inhibitor pretreatment group (PCSK9 inhibitor+LPS group) and PCSK9 inhibitor control group (PCSK9 inhibitor group), with 20 mice in each group. The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg, and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline. PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline, respectively, and the control group and LPS group were injected with an equal amount of sterile normal saline. The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling. Blood was taken from the heart for determining PLT. Platelet activation was detected by flow cytometry. The expression level of platelet-activation marker CD40L was detected by Western blotting.Results:① Clinical trial: there were 57 cases in the sepsis group and 27 cases in the healthy control group. Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group (μg/L: 232.25±72.21 vs. 191.72±54.92, P < 0.05), and PLT was significantly lower than that in the healthy control group [×10 9/L: 146.00 (75.50, 204.50) vs. 224.00 (194.00, 247.00), P < 0.01]. Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients ( n = 20) was significantly higher than that in the non-thrombocytopenia patients ( n = 37; μg/L: 264.04±60.40 vs. 215.06±72.95, P < 0.01). Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients ( r = -0.340, P = 0.010). ② Animal experiment: there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope, and no significant differences in PLT, platelet activation and plasma CD40L protein expression was found between the two groups. In the LPS group, a large number of inflammatory cells were infiltrated in the pulmonary interstitium, the alveolar structure was damaged obviously, the alveolar septum was widened, the alveolar cavity was extensively bleeding, the capillary dilatation with bleeding and platelet aggregation were found, the PLT was significantly decreased, the platelet activation and the expression level of CD40L protein in plasma were significantly increased. The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent, the thickening of alveolar septa was reduced, the platelet aggregation in lung tissue was decreased as compared with the LPS group, the PLT was significantly increased (×10 9/L: 515.83±46.60 vs. 324.83±46.31, P < 0.05), the platelet activation and the expression level of CD40L protein in plasma were significantly decreased [positive expression rate of platelet activation dependent granule surface facial mask protein CD62P: (12.15±1.39)% vs. (18.33±2.74)%, CD40L protein (CD40L/β-actin): 0.77±0.08 vs. 1.18±0.10, both P < 0.05]. Conclusion:PCSK9 level has a certain effect on promoting platelet activation in sepsis, and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.
6.Analysis of the effect of stent-assisted embolization for low-grade subarachnoid hemorrhage caused by V4 segment dissecting aneurysm of vertebral artery
Fangyu YANG ; Jiaming LIU ; Xu GAO ; Haifeng ZHANG ; Yushu DONG ; Jun LIU ; Minghao ZHOU ; Guobiao LIANG
Chinese Journal of Surgery 2021;59(8):691-696
Objective:To investigate the efficacy and safety of different stents assisted embolization in the treatment of subarachnoid hemorrhage(SAH) caused by V4 dissecting aneurysm of vertebral artery.Methods:The clinical data of 39 patients with spontaneous SAH V4 dissecting aneurysm treated at the Department of Neurosurgery, the Northern Theater General Hospital from January 2016 to June 2019 were analyzed retrospectively.There were 21 males and 18 females, aged (48±17) years(range:35 to 68 years).There were 24 cases of HUNT-HESS grade Ⅰ and 15 cases of grade Ⅱ.Among them, 20 cases were treated with single stent-assisted embolization, 9 cases with multi-stent-assisted embolization, 9 cases with semi-dense mesh-assisted embolization, and 1 case with dense-mesh stent-assisted embolization.The perioperative and postoperative complications, postoperative recurrence were collected.Results:Intraoperative complications included 2 cases of aneurysm rupture and 2 cases of acute thrombosis.All aneurysms were densely packed according to the angiography performed immediately after operation.Postoperative complications included 3 cases of long-term responsible vascular ischemia(modified Rankin score<2). The patients were followed up for 15.1 months(range: 12 to 29 months). At the last follow-up, aneurysms recurrence occured in 10 cases, the recurrence rate was 25.6%(10/39). There were 6 cases of recurrence and 2 cases of complications in 20 cases with single stent-assisted embolization, 3 cases of recurrence and 4 cases of complications in 9 cases with multi-stent-assisted embolization, 1 case of recurrence and 1 case of complications in 9 cases with semi-dense mesh stent.Conclusion:Endovascular treatment is feasible for patients with vertebral artery dissecting aneurysm, and the appropriate surgical method should be selected according to the vascular structure and the location of the aneurysm.
7.Analysis of the effect of stent-assisted embolization for low-grade subarachnoid hemorrhage caused by V4 segment dissecting aneurysm of vertebral artery
Fangyu YANG ; Jiaming LIU ; Xu GAO ; Haifeng ZHANG ; Yushu DONG ; Jun LIU ; Minghao ZHOU ; Guobiao LIANG
Chinese Journal of Surgery 2021;59(8):691-696
Objective:To investigate the efficacy and safety of different stents assisted embolization in the treatment of subarachnoid hemorrhage(SAH) caused by V4 dissecting aneurysm of vertebral artery.Methods:The clinical data of 39 patients with spontaneous SAH V4 dissecting aneurysm treated at the Department of Neurosurgery, the Northern Theater General Hospital from January 2016 to June 2019 were analyzed retrospectively.There were 21 males and 18 females, aged (48±17) years(range:35 to 68 years).There were 24 cases of HUNT-HESS grade Ⅰ and 15 cases of grade Ⅱ.Among them, 20 cases were treated with single stent-assisted embolization, 9 cases with multi-stent-assisted embolization, 9 cases with semi-dense mesh-assisted embolization, and 1 case with dense-mesh stent-assisted embolization.The perioperative and postoperative complications, postoperative recurrence were collected.Results:Intraoperative complications included 2 cases of aneurysm rupture and 2 cases of acute thrombosis.All aneurysms were densely packed according to the angiography performed immediately after operation.Postoperative complications included 3 cases of long-term responsible vascular ischemia(modified Rankin score<2). The patients were followed up for 15.1 months(range: 12 to 29 months). At the last follow-up, aneurysms recurrence occured in 10 cases, the recurrence rate was 25.6%(10/39). There were 6 cases of recurrence and 2 cases of complications in 20 cases with single stent-assisted embolization, 3 cases of recurrence and 4 cases of complications in 9 cases with multi-stent-assisted embolization, 1 case of recurrence and 1 case of complications in 9 cases with semi-dense mesh stent.Conclusion:Endovascular treatment is feasible for patients with vertebral artery dissecting aneurysm, and the appropriate surgical method should be selected according to the vascular structure and the location of the aneurysm.
8.Toddalia asiatica attnuates cerebral ischemia-reperfusion injury in rats through modulating M1/M2 polarization of microglia
Jianhong GAO ; Gang WANG ; Dan YANG ; Fangyu ZHAO ; Yiduo HE ; Xian-Bing CHEN
Chinese Journal of Pathophysiology 2023;39(12):2133-2140
AIM:Observation of neuroprotective effects of Toddalia asiatica(TA)on cerebral ischemia-reper-fusion injury(CIRI)in rats by investigating the effects and mechanisms of drugs on the polarization of microglia M1/M2 subtype and the Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB)path-way.METHODS:The modified thread occlusion method was used to establish a rat model of CIRI,and the rats were ran-domly divided into the model group,Toddalia asiatica(1.08 g/kg)group,donepezil hydrochloride(0.45 mg/kg)group,and sham group,with 16 rats in each group.Based on the assessment of neurofunctional changes in each group of rats,HE and Nissl staining were used to observe the pathological changes in brain tissue.TUNEL staining was performed to de-tect neuronal apoptosis,immunohistochemistry was used to detect the expression of M1 microglia marker ionized calcium-binding adapter molecule 1(Iba1),M2 microglia marker arginase 1(Arg1),and TLR4 localization.Western blot was used to detect the expression of microglia polarization proteins and proteins related to TLR4/MyD88/NF-κB pathway in the hippocampus region.RESULTS:Compared with sham group,the model group rats had higher neurological function scores(P<0.01),and neuronal arrangement in the hippocampus and cortex was loose and disordered,Nissl bodies de-creased,and neuronal apoptosis increased.The numbers of M1 microglia marker Iba1-,M2 microglia marker Arg1-,and TLR4-positive cells were significantly increased.In addition,the protein levels of TLR4,MyD88,p-NF-κB p65,NF-κB p65,p-NF-κB inhibitory factor(p-IκB),Iba1,interleukin-6(IL-6),and Arg1 in the hippocampus were elevated(P<0.05),while IL-4 and IL-10 expression were decreased(P<0.01).Compared with model group,the Toddalia asiatica group and Donepezil hydrochloride group showed increased protein expression of Arg1,IL-4 and IL-10(P<0.05),while the other indicators were decreased(P<0.05,P<0.01).CONCLUSION:Toddalia asiatica possesses neuroprotective effects on CIRI rats,which may be attributed to its ability to regulate M1/M2 polarization and inhibit the TLR4/MyD88/NF-κB-mediated inflammatory pathway.
9.Study on the Mechanism of Liuling Jiedu Pills Inhibiting Acute Pharyngitis Induced by Staphylococcus Aureus in Rats
Yuyan GAO ; Jinfu LU ; Gaohong LYU ; Hengbin WANG ; Niancui LUO ; Fangyu YANG ; Huiqin XU ; Bin YU ; Liuqing DI ; Ping DU
Traditional Chinese Drug Research & Clinical Pharmacology 2023;34(12):1736-1742
Objective To investigate the effect and mechanism of Liuling Jiedu Pills on acute pharyngitis caused by Staphylococcus aureus in rats.Methods The rat model of acute pharyngitis was replicated using the method of injecting 1×109 CFU·mL-1 of Staphylococcus aureus solution into the pharynx of rats.SD rats were randomly divided into a blank group,a model group,a Lanqin Oral Solution group(5 mL·kg-1),and a low-,medium-,and high-dose group of Liuling Jiedu Pills(4.375,8.750,and 17.500 mg·kg-1),with 10 rats in each group.Rats in each group were administered the drug by gavage once a day for 7 days.The general conditions of the rats were observed and recorded every day during the modeling and drug administration periods,and the local inflammation in the pharynx was scored;histopathological changes in the pharynx of the rats were observed by hematoxylin-eosin(HE)staining;serum interleukin 1β(IL-1β),interleukin 6(IL-6),tumor necrosis factor α(TNF-α),and tumor necrosis factor-α(TNF-α)were detected by ELISA.Immunohistochemistry and Western Blot were used to detect the protein expression levels of IL-1β,IL-6 and TNF-α in rat pharyngeal tissue.Results Compared with the blank group,rats in the model group had significantly increased pharyngeal erythema,significantly higher inflammation scores(P<0.01),significantly lower body mass on days 5-7 after modeling(P<0.05,P<0.01),significantly higher pathological scores(P<0.01),significantly higher levels of the serum inflammatory factors IL-1β,IL-6,and TNF-α(P<0.01),and significantly higher pharyngeal tissues showed significantly higher levels of IL-1β,IL-6,and TNF-α proteins(P<0.01).Compared with the model group,the pharyngeal erythema was significantly reduced in the Lanqin Oral Solution group and the low-,medium-and high-dose groups of Liuling Jiedu Pills,and the inflammation scores were significantly reduced(P<0.01),and the serum levels of IL-1β,IL-6,and TNF-α were significantly reduced(P<0.01);the body mass of the rats in the Lanqin Oral Solution group,and in the medium-and high-dose groups of Liuling Jiedu Pills,were significantly increased on the seventh day of the modeling(P<0.01);the histopathological scores and the levels of IL-1β,IL-6 and TNF-α proteins in pharyngeal tissue were significantly decreased(P<0.05,P<0.01).Conclusion Liuling Jiedu Pills can significantly improve the symptoms and inflammatory pathological changes of pharyngeal tissues in rats with acute pharyngitis,and its mechanism may be related to the down-regulation of the expression levels of inflammatory factors such as IL-1β,IL-6,and TNF-α.
10.Exploration on the Mechanism of Ganmao Qingre Pills Against Lung Injury Based on Network Pharmacology,Molecular Docking and Experimental Verification
Wei REN ; Mingming XU ; Xin GAO ; Bowen MA ; Ziren SU ; Yuhong LIU ; Fangyu ZHAO
Traditional Chinese Drug Research & Clinical Pharmacology 2024;35(9):1376-1388
Objective To investigate the mechanism of Ganmao Qingre Pills(GQP)against lung injury based on network pharmacology,molecular docking and in vivo experiments.Methods The potential targets of GQP in the treatment of lung injury were screened through traditional Chinese medicine systems pharmacology database and analysis platform(TCM-SP)and Genecards.A"Chinese medicine-active ingredients-targets"network was constructed using Cytoscape 3.9.0 software,then gene ontology(GO)function and Kyoto encyclopaedia of genes and genomes(KEGG)pathway enrichment analysis for potential targets were conducted using a bioinformatics cloud platform.We established a protein-protein interaction(PPI)network,which was intersected with"Chinese medicine-active ingredients-targets"network to obtain core targets.The molecular docking between key target proteins and active ingredients was performed.The effect of GQP on these key target proteins was verified by using a mouse model of lung injury.Results A total of 707 targets for the treatment of lung injury by GQP were identified,corresponding to 107 active ingredients in 11 Chinese medicines.It was found that GQP might regulate targets such as PTGS1,AR,and ACHE through active ingredients including stigmasterol,luteolin,and acacetin using the"Chinese medicine-active ingredients-targets"network analysis.Core targets such as SRC,EGFR,and STAT3 were discovered by using the PPI network.Key target proteins,including CDK1,CDK2,EGFR,ESR1 and SRC,were screened through the intersection analysis of the PPI network and"Chinese medicine-active ingredients-targets"network.Molecular docking study showed that stigmasterol,luteolin and acacetin had good binding effects with CDK1,CDK2,EGFR,ESR1,and SRC,respectively.In vivo experiments revealed that GQP dose-dependently attenuated lung injury and inflammatory infiltration,reduced the release of pro-inflammatory factors TNF-α,IL-1β and IL-6,increased the expression of CDK1 and CDK2,and decreased the expression of EGFR,ESR1 and SRC in lung injury mice.Conclusion The therapeutic effect of GQP against lung injury may be achieved through interaction of key active ingredients(stigmasterol,luteolin,and acacetin)and key target proteins(CDK1,CDK2,EGFR,ESR1,SRC),and regulation of key signaling pathways such as neuroactive ligand-receptor interactions,cancer pathways,and calcium signaling pathways.