ObjectiveTo observe the effects of Qingxin Jieyu granules （QXJYG） on FeCl₃-induced carotid artery thrombosis in rats and on the expression of thrombosis-related proteins tissue factor （TF） and tissue factor pathway inhibitor （TFPI） as well as the protein kinase B （Akt）/nuclear factor-κB （NF-κB） signaling pathway in EA.hy926 cells exposed to tumor necrosis factor-α （TNF-α）， thus preliminarily exploring the mechanism of QXJYG in inhibiting thrombosis. MethodsThirty-six SD rats were randomized into normal control， model， positive control （aspirin， 9 mg·kg^-1）， and low-， medium-， and high-dose （0.99， 1.98， 3.96 g·kg^-1， respectively） QXJYG groups （n=6）. The rats in the drug treatment groups were administrated with corresponding drugs， and those in the normal control group and model group were given an equal volume of distilled water. After 14 consecutive days of prophylactic gavage， the rat model of common carotid artery thrombosis was established with 45% FeCl₃ solution， and the blood vessels were collected and the wet weight of thrombus was weighed by an electronic balance （precision of 1/10 000）. The thrombosis in the common carotid artery of each group of rats was observed by hematoxylin-eosin staining. The plasma levels of von Willebrand factor （vWF）， platelet endothelial cell adhesion molecule-1 （PECAM-1）， tissue-type plasminogen activator （t-PA）， and plasminogen activator inhibitor-1 （PAI-1） were determined by enzyme-linked immunosorbent assay. An endothelial cell injury model was established by treating EA.hy926 human umbilical vein endothelial cells with TNF-α. The cell counting kit-8 method was used to screen the intervention concentrations of QXJYG. Western blot was employed to determine the protein levels of TF， TFPI， Akt， p-Akt， NF-κB p65， and p-NF-κB p65 in each group of cells. ResultsThe animal experiment showed that compared with the normal control group， the model group showed an increase in carotid artery thrombus weight （P<0.05）， with unclear vascular structure and extensive thrombosis in the lumen. In addition， the plasma levels of vWF， PECAM-1， and PAI-1 were elevated， while the t-PA level became lowered （P<0.05） in the model group. Compared with the model group， the aspirin and QXJYG groups showed reductions in the weight of FeCl₃-induced carotid artery thrombi （P<0.05） and thrombosis in the lumen， declines in plasma levels of PECAM-1 and PAI-1， and an elevation in the t-PA level （P<0.05）. Moreover， the QXJYG groups showed reductions in the plasma level of vWF （P<0.05）， which， however， had no significant difference between the aspirin group and the model group. The cell experiments indicated that 31.25， 62.5， 125， 250， 500 mg·L^-1 QXJYG had no effect on the viability of EA.hy926 cells. Therefore， 250， 500 mg·L^-1 QXJYG were selected as the intervention concentrations for subsequent experiments. Western blotting results showed that compared with the control group， the TNF-α stimulation downregulated the expression of TFPI （P<0.05）， upregulated the expression of TF， and increased the ratios of p-Akt/Akt and p-NF-κB p65/NF-κB p65 （P<0.05） in EA.hy926 cells. Compared with the model group， the intervention with QXJYG upregulated the expression of TFPI （P<0.05）， inhibited the expression of TF， and decreased the ratios of p-Akt/Akt and p-NF-κB p65/NF-κB p65 （P<0.05）. ConclusionQXJYG has the effect of inhibiting thrombosis and regulating the expression of TF and TFPI in endothelial cells exposed to TNF-α by suppressing the abnormal activation of the Akt/NF-κB signaling pathway.

Objective To observe the effect of Qing-Xin-Jie-Yu granule(QXJYG)on the formation of thrombosis in the rat model of arteriovenous bypass thrombosis and the adenosine diphosphate(ADP)-induced platelet aggregation.Methods Thirty-six male SD rats were randomly divided into normal control group,model group,clopidogrel positive control group,QXJYG low-dose,medium-dose and high-dose groups,with 6 rats in each group.The dose of clopidogrel positive control group was 6.74 mg/(kg?d),the dosages of QXJYG in low,medium and high groups were 0.99,1.98,3.96 g/(kg?d),respectively,normal control group and model group were given equal volume of distilled water,and continuous prophylactic intragastric administration for 14 days,once a day.One hour after the final administration,the rats were anesthetized,and the arteriovenous bypass thrombosis model was established by using a polyethylene tube as the arteriovenous bypass bridge(except control group).The thrombus was extracted after 15 min and its weight was weighed by 1/10,000th precision electronic balance.The levels of thromboxane B2(TXB2)and 6-keto-prostaglandin F1α(6-keto-PGF1α)in plasma were determined by ELISA kits.The rate of platelet aggregation induced by ADP in each group was measured using a microplate reader by turbidimetric method.Results Compared with the control group,the weight of arteriovenous bypass thrombus was significantly higher,the level of TXB2 in plasma was significantly higher,while the level of 6-keto-PGF1α was significantly lower,and platelet aggregation was significantly higher after ADP induction in the model group(P＜0.05).Compared with the model group,the weight of arteriovenous bypass thrombosis in clopidogrel positive control group and QXJYG dose groups was significantly decreased(P＜0.05);the inhibition rate of thrombosis formation was 53.80％,23.96％,33.63％,and 32.59％,respectively.The content of TXB2 in plasma was significantly decreased,the content of 6-keto-PGF1α was significantly increased;additionally,the platelet aggregation rate induced by ADP was reduced in clopidogrel positive control group and QXJYG group.Meanwhile,there was a dose-dependence between different doses in QXJYY group(P＜0.05),and the inhibition rate of platelet aggregation was 86.90％,26.17％,38.87％,54.48％,respectively.Conclusion QXJYG can prevent thrombosis formation in the rat model of arteriovenous bypass thrombosis and inhibit platelet aggregation induced by ADP.

Objective To observe the effect of Qing-Xin-Jie-Yu granule(QXJYG)on the formation of thrombosis in the rat model of arteriovenous bypass thrombosis and the adenosine diphosphate(ADP)-induced platelet aggregation.Methods Thirty-six male SD rats were randomly divided into normal control group,model group,clopidogrel positive control group,QXJYG low-dose,medium-dose and high-dose groups,with 6 rats in each group.The dose of clopidogrel positive control group was 6.74 mg/(kg?d),the dosages of QXJYG in low,medium and high groups were 0.99,1.98,3.96 g/(kg?d),respectively,normal control group and model group were given equal volume of distilled water,and continuous prophylactic intragastric administration for 14 days,once a day.One hour after the final administration,the rats were anesthetized,and the arteriovenous bypass thrombosis model was established by using a polyethylene tube as the arteriovenous bypass bridge(except control group).The thrombus was extracted after 15 min and its weight was weighed by 1/10,000th precision electronic balance.The levels of thromboxane B2(TXB2)and 6-keto-prostaglandin F1α(6-keto-PGF1α)in plasma were determined by ELISA kits.The rate of platelet aggregation induced by ADP in each group was measured using a microplate reader by turbidimetric method.Results Compared with the control group,the weight of arteriovenous bypass thrombus was significantly higher,the level of TXB2 in plasma was significantly higher,while the level of 6-keto-PGF1α was significantly lower,and platelet aggregation was significantly higher after ADP induction in the model group(P＜0.05).Compared with the model group,the weight of arteriovenous bypass thrombosis in clopidogrel positive control group and QXJYG dose groups was significantly decreased(P＜0.05);the inhibition rate of thrombosis formation was 53.80％,23.96％,33.63％,and 32.59％,respectively.The content of TXB2 in plasma was significantly decreased,the content of 6-keto-PGF1α was significantly increased;additionally,the platelet aggregation rate induced by ADP was reduced in clopidogrel positive control group and QXJYG group.Meanwhile,there was a dose-dependence between different doses in QXJYY group(P＜0.05),and the inhibition rate of platelet aggregation was 86.90％,26.17％,38.87％,54.48％,respectively.Conclusion QXJYG can prevent thrombosis formation in the rat model of arteriovenous bypass thrombosis and inhibit platelet aggregation induced by ADP.

Objective To elucidate the therapeutic mechanism of Qingxin Jieyu Granule(QXJYG)against atherosclerosis(AS)based on network pharmacology.Methods The major targets and pathways of QXJYG against AS were analyzed using network pharmacology.Rat models of AS established by high-fat feeding combined with intraperitoneal vitamin D3 injection were treated daily with normal saline,atorvastatin(13.15 mg/kg),or QXJYG at 0.99,1.98,and 3.96 g/kg for 8 weeks(n=6).Ultrasound and HE staining were used to assess the function and pathologies of the abdominal aorta.Blood lipids and serum levels of Ang II,ET-1,TXA2,PGI2,and ox-LDL of the rats were detected using an automatic biochemical analyzer or ELISA.The expressions of LOX-1,PPARγ,RXRα,p-P65,VCAM-1 and ICAM-1 in the abdominal aorta were detected with immunohistochemistry.Results The rat models of AS showed obvious abdominal aorta wall thickening,increased pulse wave velocity and pulse index,decreased inner diameter of the abdominal aorta,elevated levels of TC,LDL-C,Ang II,ET-1 and TXA2,and lowered levels of HDL-C and PGI2.QXJYG and atorvastatin treatment of the rat models significantly alleviated histopathological changes of the abdominal aorta,decreased serum levels of TC,LDL-C,Ang II,ET-1 and TXA2,and increased the levels of HDL-C and PGI2.Network pharmacology study suggested the therapeutic effect of QXJYG against AS was mediated by regulating lipid metabolism,PPAR and NF-κB pathways.Consistently,treatments with QXJYG were found to significantly decrease ox-LDL level and LOX-1,P-P65,VCAM-1 and ICAM-1 protein expressions while increasing PPARγ and RXRα expressions in the aorta of AS rats.Conclusion QXJYG alleviates lipid metabolism disorder and improves histopathological changes of the abdominal aorta of AS rats possibly by lowering ox-LDL level,reducing LOX-1 expression,activating PPARγ and RXRα,and inhibiting P65 phosphorylation to reduce VCAM-1 and ICAM-1 expression in the aorta.

Objective To elucidate the therapeutic mechanism of Qingxin Jieyu Granule(QXJYG)against atherosclerosis(AS)based on network pharmacology.Methods The major targets and pathways of QXJYG against AS were analyzed using network pharmacology.Rat models of AS established by high-fat feeding combined with intraperitoneal vitamin D3 injection were treated daily with normal saline,atorvastatin(13.15 mg/kg),or QXJYG at 0.99,1.98,and 3.96 g/kg for 8 weeks(n=6).Ultrasound and HE staining were used to assess the function and pathologies of the abdominal aorta.Blood lipids and serum levels of Ang II,ET-1,TXA2,PGI2,and ox-LDL of the rats were detected using an automatic biochemical analyzer or ELISA.The expressions of LOX-1,PPARγ,RXRα,p-P65,VCAM-1 and ICAM-1 in the abdominal aorta were detected with immunohistochemistry.Results The rat models of AS showed obvious abdominal aorta wall thickening,increased pulse wave velocity and pulse index,decreased inner diameter of the abdominal aorta,elevated levels of TC,LDL-C,Ang II,ET-1 and TXA2,and lowered levels of HDL-C and PGI2.QXJYG and atorvastatin treatment of the rat models significantly alleviated histopathological changes of the abdominal aorta,decreased serum levels of TC,LDL-C,Ang II,ET-1 and TXA2,and increased the levels of HDL-C and PGI2.Network pharmacology study suggested the therapeutic effect of QXJYG against AS was mediated by regulating lipid metabolism,PPAR and NF-κB pathways.Consistently,treatments with QXJYG were found to significantly decrease ox-LDL level and LOX-1,P-P65,VCAM-1 and ICAM-1 protein expressions while increasing PPARγ and RXRα expressions in the aorta of AS rats.Conclusion QXJYG alleviates lipid metabolism disorder and improves histopathological changes of the abdominal aorta of AS rats possibly by lowering ox-LDL level,reducing LOX-1 expression,activating PPARγ and RXRα,and inhibiting P65 phosphorylation to reduce VCAM-1 and ICAM-1 expression in the aorta.

OB JECTIVE To study the anti-inflammatory effect of couplet medicinals of Achyranthes bidentata -Eucommia ulmoides. METHODS Mouse macrophage RAW 264.7 were divided into blank group ，model group ，A. bidentata group（800 μg/mL），E. ulmoides group（800 μg/mL）and low- ，medium- and high- concentration groups of couplet medicinals of A. bidentata - E. ulmoides （400，800，1 600 μg/mL）. Excep for blank group and model group ，the other groups were added with corresponding drugs for 6 hours；then blank group was continued to add into the medium ，while model group was added into 10 μg/mL lipopolysaccharide （to induce the inflammatory model ）；other groups were added into corresponding drugs and 10 μ g/mL lipopolysaccharide. The levels of inflammatory factors [nitric oxide （NO），interleukin-1β（IL-1β），IL-6，tumor necrosis factor-α （TNF-α）] were detected ，and Jin ’s formula was used to evaluate the effects of A. bidentata -E. ulmoides . The expression of inducible nitric oxide synthase （iNOS），cyclooxygenase-2（COX-2），nuclear factor kappa-B （NF-κB）and inhibitor α of NF-κB （IκBα）as well as the phosphorylation of NF-κB p65，IκB kinase（IKK），p38 mitogen-activated protein kinase （p38 MAPK）， extracellular signal-regulated kinase （ERK）and c-Jun N-terminal kinase （JNK）were determined. RESULTS Compared with blank group，the level of inflammatory factors ，protein expression of iNOS and COX- 2 as well as the phosphorylation of NF-κB p65， IKK，p38 MAPK，ERK and JNK were increased significantly （P＜0.01），while the protein expression of IκBα was decreased significantly（P＜0.01）. After intervention of couplet medicinals of A. bidentata -E. ulmoides ，the level of inflammatory factors ，the expression or phosphorylation of above proteins were reversed significantly （P＜0.05 or P＜0.01），and couplet medicinals of A. bidentata-E. ulmoides had a synergistic effect. CONCLUSIONS The couplet medicinals of A. bidentata -E. ulmoides have synergistic anti-inflammatory effect on RAW 264.7 cells. Its mechanism may be related to the inhibition of NF-κB/MAPK signaling pathway related protein expression.

Objective:To evaluate the clinical efficacy of Wanfu-Qutong Decoction combined with esomeprazole in the treatment of chronic atrophic gastritis (CAG). Methods:A total of 106 CAG patients who met the inclusion criteria from June 2017 to June 2019 were randomly divided into two groups with 53 in each group. The control group took esomeprazole magnesium enteric coated tablets, and the observation group took Wanfu-Qutong Decoction on the basis of the control group. Both groups were treated continuously for 3 months. TCM syndrome score was performed before and after treatment, and the new Sydney system intuitive simulation score method was used to score the histopathology of gastric mucosa. The levels of gastrin 17 (G-17), pepsinogen (PGⅠ , PGⅡ) and the PG Ⅰ/Ⅱ were measured by ELISA. Results:The total effective rate was 96.2% (51/53) in the observation group and 79.2% (42/53) in the control group. There was significant difference between the two groups ( χ2=7.414, P<0.01). After treatment, the scores of epigastric pain, fullness, liking temperature and pressing, vomiting clear water, eating less and staying foolish, and limb burnout in the observation group were significantly lower than those in the control group ( t values were 2.788, 3.632, 3.816, 1.590, 2.183, 2.103, respectively, all Ps<0.05), and the scores of chronic inflammatory reaction, inflammatory activity, atrophy degree, dysplasia and intestinal metaplasia in the mucosa were significantly lower than those in the control group ( t values were 2.983, 2.106, 2.106, 3.773, 1.922, 3.095, respectively, all Ps<0.05). After treatment, the serum G-17 [(14.47 ± 3.06) pmol/L vs. (10.67 ± 2.47) pmol/L, t=10.510] and PG Ⅰ [(130.31 ± 14.79) μg/L vs. (102.36 ± 12.63) μg/L, t=8.178] and PG Ⅰ/Ⅱ [(10.45 ± 0.48) vs. (9.17 ± 0.72), t=2.104] in the observation group were significantly higher than those in the control group ( P<0.01 or P<0.05). Conclusion:Wanfu-Qutong Decoction combined with esomeprazole tablets can effectively improve the clinical symptoms of CAG patients, regulate the levels of G-17, PG Ⅰ , PG Ⅱ and PGⅠ/Ⅱ, and promote the repair of gastric mucosa.

Objective To explore the effects of referral and rehabilitation status of patients with total knee arthroplasty (TKA) under the mode of medical alliance.Methods One hundred and ninety cases with total knee arthroplasty in the secondary rehabilitation hospitals after referral from a tertiary hospital from January 2016 to January 2017 were analyzed, including general demographic data, disease and surgical data, rehabilitation data after referral, hospital day and change of medical expenses of the tertiary hospital and rehabilitation hospitals before and after the promotion of the medical alliance mode.Results After receiving the rehabilitation treatment in secondary rehabilitation hospitals, the range of motion, muscle strength and some other indices of the patients' surgical legs were significantly improved (P<0.05). And the hospital day of the surgical hospital was shorten from (15.05±6.59) d to (12.97±5.24) d, and medical expenses were decreased by 2.18% in total, 4.76% per knee and 14.01% in rehabilitation costs.Conclusions Under the mode of medical alliance, transferring patients with TKA from a tertiary hospital to secondary rehabilitation hospitals in their early postoperative phase can facilitate recovery of the patients, and it can also help to reduce hospital day and medical expenses, thus to optimize the allocation of medical resources.

Objective To introduce a practical method that can be used to efficiently express,purify and identify Alzheimer's disease (AD) related beta-site app-cleaving enzyme 1 (BACE1) in common eukaryotic cells.Methods BACE1 cDNA was fished out from human brain cDNA library and ligated into the pEGFP-c3 expression vector,and then,the recombinant plasmid was transfected into the HEK293 cells.The BACE1 protein was purified with TALON Mental Affinity Resins column.The target protein was identified by Western blotting and fluorescence resonance energy transfer (FRET).BACE1 Activity Assay Kit was employed to test the activity of purified BACE1 in vitro.The recombinant BACE1/pEGFP-c3 plasmid and amyloid precusor protein (APP)/pDsRed-Monomer-N1 plasmid were co-transfected to the HEK293 cells and the cleavage activity of BACE1 in the cells was identified by Western blotting.Results The sequencing data of the obtained BACE1 gene were identical with those in GenBank.Activity test showed that the fluorescent values of blank controls,expressed BACE1 and standard BACE1 were 55.013±3.597,1836.629±154.195 (n=3) and 2639.548±207.1901 (n=3),respectively;as compared with the control group,significant differences were noted in both of the two groups (F=78.681,P=0.000);however,there is no significant difference between expressed BACE1 and standard BACE1 groups (P＞0.05).Westem blotting showed the co-transfected BACE1 could cleave APP in HEK293 cells and the CTF-APP band was detectable.Conclusion A practical protocol is established for high expression,purification and identification of BACE1 in HEK293 cells,which is helpful to obtain BACE1,an important molecular target in AD research and treatment.

Objective:To predict the secondary structure and B-cell epitope of human IL-37.Methods:Based on IL-37b ami-no acid sequence, the secondary structure was predicted by SOPMA; hydrophilicity, flexibility, accessibility index were predicted by software of ProScale, Bcepred, respectively.Combined the results according to these methods , the B cell epitopes of IL-37b were pre-dicted.Results: The second structure of IL-37b contained extended strand (31.65%), random coil (52.75%), alpha helix (8.26%), beta turn (7.34%) and the most possible epitopes of IL-37b were located in or adjacent to amino acid 21-27, 34-75, 175-192 , 213-215 .Conclusion:These results will be helpful for the estimate of the epitopes and provide a theory basis for developing mon -oclonal antibodies against human IL-37.