Protective Effect of Qihong Tongluo Prescription on Vascular Endothelial Cells in Rats with Deep Venous Thrombosis Based on NF-κB Pathway / 中国实验方剂学杂志

ObjectiveTo explore the protective effect and mechanism of Qihong Tongluo prescription on vascular endothelial cells in rats with deep venous thrombosis （DVT）. MethodSixty-six SD rats were randomly divided into a blank group （n=11） and a modeling group （n=55）. The DVT model was induced in rats of the modeling group by slowing down blood flow and damaging vascular endothelium. The model rats were randomly divided into model group， aspirin group （200 mg·kg-1）， and low-，medium-， and high-dose Qihong Tongluo prescription groups （6.5， 13， 26 g·kg-1） according to a random number table. Rats were treated with corresponding drugs by gavage， while those in the model group and the blank group received normal saline， once per day for 7 days. The rats were sacrificed and the abdominal aortic blood was taken. The levels of serum endothelin-1 （ET-1） and interleukin-6 （IL-6） were detected by enzyme-linked immunosorbent assay （ELISA）. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in vascular endothelial tissues. The ultrastructure of vascular endothelial cells was observed by the transmission electron microscope. The viability of vascular endothelial cells was detected by methylthiazolyldiphenyl-tetrazolium bromide （MTT） method，and the release level of lactate dehydrogenase （LDH） was detected by the LDH kit. The messenger ribonucleic acid （mRNA） expression of platelet-activating factor （PAF），nuclear transcription factor κB （NF-κB），Ras-related C3 botulinum toxin substrate 1 （Rac1）， and Ras-related C3 botulinum toxin substrate 2 （Rac2） in vascular endothelial tissues were detected by real-time reverse transcription polymerase chain reaction （Real-time PCR）. The protein expression of PAF，NF-κB，Rac1， and Rac2 in vascular endothelial tissues was detected by Western blot. ResultThe model group showed seriously damaged and swollen vascular endothelial cells with massive shedding， attachment of massive inflammatory cells， nucleus pyknosis and deformation under the electron microscope， highly swollen mitochondria， serious cytoplasmic vacuolation，and exposure of internal elastic membrane. The damage of vascular endothelium and its ultrastructure in Qihong Tongluo prescription groups and the aspirin group was improved in varying degrees. Compared with the blank group，the model group showed increased levels of serum ET-1 and IL-6，potentiated vascular endothelial cell viability， up-regulated mRNA and protein expression of PAF，NF-κB，Rac1， and Rac2 in vascular endothelial tissues，and decreased LDH release level of vascular endothelial cells （P<0.05）. Compared with the model group，the aspirin group and the Qihong Tongluo prescription groups showed decreased levels of serum ET-1 and IL-6，blunted vascular endothelial cell viability，down-regulated mRNA and protein expression of PAF，NF-κB，Rac1， and Rac2 in vascular endothelial tissues，and increased LDH release level of vascular endothelial cells （P<0.05）. The effect of Qihong Tongluo prescription was dose-dependent. ConclusionQihong Tongluo prescription has a protective effect on vascular endothelial cells of DVT rats and can prevent and treat thrombosis，and its therapeutic effect is presumably achieved by inhibiting the expression of PAF，NF-κB，Rac1，and Rac2 and reducing the levels of serum ET-1 and IL-6.

Single cell sequencing analysis uncovers genetics-influenced CD16+monocytes and memory CD8+T cells involved in severe COVID-19

BackgroundUnderstanding the host genetic architecture and viral immunity contributes to the development of effective vaccines and therapeutics for controlling the COVID-19 pandemic. Alterations of immune responses in peripheral blood mononuclear cells play a crucial role in the detrimental progression of COVID-19. However, the effects of host genetic factors on immune responses for severe COVID-19 remain largely unknown. MethodsWe constructed a powerful computational framework to characterize the host genetics-influenced immune cell subpopulations for severe COVID-19 by integrating GWAS summary statistics (N = 969,689 samples) with four independent scRNA-seq datasets (N = 606,534 cells). ResultsWe found that 34 risk genes were significantly associated with severe COVID-19, and the number of highly-expressed genetics-risk genes increased with the severity of COVID-19. Three cell-subtypes that are CD16+monocytes, megakaryocytes, and memory CD8+T cells were significantly enriched by COVID-19-related genetic association signals. Notably, three causal risk genes of CCR1, CXCR6, and ABO were specifically expressed in these three cell types, respectively. CCR1+CD16+monocytes and ABO+ megakaryocytes with significant up-regulated genes including S100A12, S100A8, S100A9, and IFITM1 confer higher risk to the cytokine storms among severe patients. CXCR6+ memory CD8+ T cells exhibit a notable polyfunctionality of multiple immunologic features, including elevation of proliferation, migration, and chemotaxis. Moreover, we observed a prominent increase in cell-cell interactions of both CCR1+ CD16+monocytes and CXCR6+ memory CD8+T cells in severe patients compared to normal controls among both PBMCs and lung tissues, and elevated interactions with epithelial cells could contribute to enhance the resident to lung airway for against COVID-19 infection. ConclusionsWe uncover a major genetics-modulated immunological shift between mild and severe infection, including an increase in up-regulated genetic-risk genes, excessive secreted inflammatory cytokines, and functional immune cell subsets contributing high risk to severity, which provides novel insights in parsing the host genetics-influenced immune cells for severe COVID-19.

COVID-19 Quarantine Reveals Grade-specific Behavioral Modification of Myopia: One-Million Chinese Schoolchildren Study

BackgroundHigh prevalence of myopia of adolescent has been a global public health concern. Their risk factors and effective prevention methods for myopia across schoolchildren developmental stages are critically needed but remain uncertain due to the difficulty in implementing intervention measurements under normal life situation. We aimed to study the impact of the COVID-19 quarantine on myopia development among over one-million schoolchildren. MethodsWe designed the ongoing longitudinal project of Myopic Epidemiology and Intervention Study (MEIS) to biannually examine myopia among millions of schoolchildren for ten years in Wenzhou City, Zhejiang Province, China. In the present study, we performed three examinations of myopia in 1,305 elementary and high schools for schoolchildren in June 2019, December 2019 and June 2020. We used the normal period (June-December 2019) and COVID-19 quarantine period (January-June 2020) for comparisons. Myopia was defined as an uncorrected visual acuity of 20/25 or less and a spherical equivalent refraction (SER) of -0.5 diopters (D) or less. High myopia was defined as an SER of -6.0 D or less. FindingsIn June 2019, 1,001,749 students aged 7-18 were eligible for examinations. In the 6-month and 12-month follow-up studies, there were 813,755 eligible students (81.2%) and 768,492 eligible students (76.7%), respectively. Among all students, we found that half-year myopia progression increased approximate 1.5 times from -0.263 D (95% CI, -0.262 to -0.264) during normal period to -0.39 D (95% CI, -0.389 to -0.391) during COVID-19 quarantine (P < 0.001). Multivariate Cox regression analysis identified grade rather than age was significantly associated with myopia (Hazard ratio [HR]: 1.10, 95% CI, 1.08 to 1.13; P < 0.001) and high myopia (HR: 1.40, 95% CI, 1.35 to 1.46; P < 0.001) after adjustment for other factors. The prevalence, progression, and incidence of myopia and high myopia could be categorized into two grade groups: I (grades 1-6) and II (grades 7-12). Specifically, COVID-19 quarantine for 6 months sufficiently increased risk of developing myopia (OR: 1.36, 95% CI, 1.33 to 1.40) or high myopia (OR: 1.30, 95% CI, 1.22 to 1.39) in Grade Group I, but decreased risk of developing myopia (OR: 0.45, 95% CI, 0.43 to 0.48) or high myopia (OR: 0.57, 95% CI, 0.54 to 0.59) in Grade Group II. InterpretationThe finding that behavioral modifications for six months during COVID-19 quarantine sufficiently and grade-specifically modify myopia development offers the largest human behavioral intervention data at the one million scale to identify the grade-specific causal factors and effective prevention methods for guiding the formulation of myopia prevention and control policies. FundingKey Program of National Natural Science Foundation of China; the National Natural Science Foundation of China; Scientific Research Foundation for Talents of Wenzhou Medical University; Key Research and Development Program of Zhejiang Province. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMyopia is the most-common refractive error worldwide. Myopia with younger onset may result in developing high myopia, which is associated with sight-threatening ocular diseases such as maculopathy, retinal detachment, opticneuropathy, glaucoma, retinal atrophy, choroidal neovascularization. In light of the increasing prevalence of myopia and high myopia has been a global public health concern, the impact of COVID-19 lockdown on myopia development has gained substantial attention. We searched PubMed, Google Scholar, and MEDLINE databases for original articles reported between database inception and November 10, 2020, using the following search terms: (coronavirus OR COVID* OR SARS-COV-2 OR lockdown OR quarantine) AND (myopia OR short-sightedness OR refractive error). To date, there was no original study reported to uncover the influence of COVID-19 quarantine on myopia progression. Added value of this studyThis study provides the largest longitudinal intervention data on myopia progression in Chinese schoolchildren covering all grades of schoolchildren at one-million scale. COVID-19 quarantine model uncovers that behavioral modifications for six months may lead to significant increase of overall prevalence of myopia associated with their increased screen times and decreased outdoor activity times. Importantly, their effects on developing myopia or high myopia of students are grade-dependent, which were risk factors for elementary schools period but protective factors for high schools period partly due to reduced school education burden. Implications of all the available evidenceThis one-million schoolchildren myopia survey offers evidence that six months behavioral modifications sufficiently and grade-specifically change the progression of myopia and high myopia. In view of the increased use of electronic devices is an unavoidable trend, effective myopia prevention strategy according to grade among students is urgently needed. Since COVID-19 outbreak is still ongoing and spreading, international collaborate efforts are warranted to uncover the influence of COVID-19 on myopia progression to further substantiate these findings.

Integrative Genomics Analysis Reveals a Novel 21q22.11 Locus Contributing to Susceptibility of COVID-19

The systematic identification of host genetic risk factors is essential for the understanding and treatment of COVID-19. By performing a meta-analysis of two independent genome-wide association (GWAS) summary datasets (N = 680,128), a novel locus at 21q22.11 was identified to be associated with COVID-19 infection (rs9976829 in IFNAR2 and upstream of IL10RB, OR = 1.16, 95% CI = 1.09 - 1.23, P = 2.57x10-6). The rs9976829 represents a strong splicing quantitative trait locus (sQTL) for both IFNAR2 and IL10RB genes, especially in lung tissue (P 1.8x10-24). Gene-based association analysis also found IFNAR2 was significantly associated with COVID-19 infection (P = 2.58x10-7). Integrative genomics analysis of combining GWAS with eQTL data showed the expression variations of IFNAR2 and IL10RB have prominent effects on COVID-19 in various types of tissues, especially in lung tissue. The majority of IFNAR2-expressing cells were dendritic cells (40%) and plasmacytoid dendritic cells (38.5%), and IL10RB-expressing cells were mainly nonclassical monocytes (29.6%). IFNAR2 and IL10RB are targeted by several interferons-related drugs. Together, our results uncover 21q22.11 as a novel susceptibility locus for COVID-19, in which individuals with G alleles of rs9976829 have a higher probability of COVID-19 susceptibility than those with non-G alleles.

Fabrication and biological effect investigation of the antibacterial drug delivery system with PEG-hydrogel and gentamicin-loaded-CSt on titanium surface / 中华骨科杂志

Objective To fabricate an antibacterial controlled drug delivery system with PEG-hydrogel and gentamicin-loaded-CSt on titanium surface,and to investigate its surface characteristics,swelling behavior,drug release behavior in vitro,antiinfection performance in vivo,and tissue biocompatibility.Methods Cross-linked starch (CSt) was synthesized first and then CSt was loaded with gentamicin (GEN) as a carrier (GEN@CSt),then 4-arm-polyethylene glycol (PEG) was added to it which was mixed by ultrasound.The surface of titanium (Ti) was covered with a layer of poly dopamine (PDA).The drug-loaded hydrogel was fixed to the titanium surface,subsequently capped by poly lactic-co-glycolic acid (PLGA) membranes,and then the Ti-PDA-PEG (GEN@CSt)-PLGA composite coating was fabricated finally.Surface morphology of the system was observed,while the swelling behavior was characterized;release behavior of the composite coating was detected;the bacteriostatic experiments were carried out with staphylococcus aureus (SAU),staphylococcus epidermidis (SEP) and escherichia coli (ECO) in vitro.The animal models of infected bone defect was established in 36 New Zealand white rabbits.These animals were randomly divided into three groups.Group 1 animals were implanted with drug-loaded composite coatings.Group 2 animals were implanted with drug-free composite coatings.Group 3 animals were implanted with bare titanium rods.The infection data were collected periodically to carry out antiinfection experiments in vivo.Another 12 rabbits were divided into the experimental group and the control group randomly.Biocompatibility of the materials was observed by histopathology after implantation of the corresponding materials into the femoral condyle.Results The composite coating adhered to the titanium surface firmly,presenting a smooth and translucent shape.The ratio of CSt/PEG affects swelling behavior varied,starch-free gels maintained an equilibrium swelling of 7.4,after the ratio reached 1 ∶ 1,the equilibrium swelling ratio remained at 3.0.In-vitro the release rate of the first 8 h was fast,and the cumulative release amount accounted for 83％ of the total in the first 7 days,lasting more than 13 d.In vitro antibacterial test,the average diameter of the inhibition ring was 3.6±0.13 cm (SAU),3.4±0.11 cm (SEP),3.7±0.10 cm (ECO).In-vivo anti-infection experiment,the infection situation of the group 1 was better than the control groups 2 and 3.The pathological results indicated that inflammatory reaction in the experimental group was basically the same as the control group.Conclusion The study successfully fabricated the antibacterial controlled drug delivery system with PEG-hydrogel and gentamicin-loaded-CSt on titanium surface.The system has a reasonable drug release behavior,and effectively inhibited the growth of bacteria in vivo and in vitro.It also has good biocompatibility to stand a promising strategy to improve the orthopedics anti-infection.

An observational study of the combination therapy of Gabapentin and Cobamamide in treatment of painful diabetic neuropathy / 中国糖尿病杂志

Objective To investigate the effect of combination therapy of Gabapentin and Cobamamide in treatment of painful diabetic neuropathy (PDN).Methods A total of 96 patients with type 2 diabetes (T2DM) and PDN were enrolled in this study and randomly divided into control group (Con,n=32),Cobamamide group,(n=32),and Gabapentin+Cobamamide group,(n=32).FPG and HbA1c were actively controlled in each group.Con group was treated with vitamin B1.Clinical and biochemical data of all the subjects were collected.The degree of pain was assessed by visual analogue scale (VAS).The changes of median nerve,peroneal nerve motor nerve conduction velocity (MNCV),and sensory nerve conduction velocity (SNCV) were evaluated by EMG assessment.The assessment of sleep quality was done by Pittsburgh sleep quality index scale (PSQI).Results There was no significant differences of baseline MNCV,SNCV and the degree of pain among the three groups (P>0.05).After treatment,all the above index were improved in both Cobamamide group and Gabapentin+Cobamamide group.MNCV and SNCV were higher in Gabapentin+Cobamamide group than in Cobamamide group (P0.05).Conclusion The combination therapy of Gabapentin and adenosine cobalt amine could reduce pain,improve nerve conduction velocity,and improve the quality of sleep.

Fabrication of the anti-tuberculosis controlled drug delivery system with Ti-PDA-PEG-PLGA-INH and investigation of the biological characteristics / 中华骨科杂志

Objective To fabricate an anti?tuberculosis controlled drug release coating with Ti?PDA?PEG?PLGA?INH and to investigate its surface characteristics, in vivo and in vitro drug release behavior, and tissue biocompatibility. Methods 4?arm?polyethylene glycol (PEG) was synthesized first. Then cover the surface of titanium (Ti) with a layer of poly dopamine (PDA) by Michael addition reaction. Use porous starch and 4?arm?PEG as a carrier, load with isoniazid (INH), then attach to the surface of titanium by casting or sol?gel dip coating methods, and then cover with a layer of poly lactic?co?glycolic acid (PLGA) by the same method, to fabricate the Ti?PDA?PEG?PLGA?INH composite coating finally. The functional group of 4?arm?PEG was charac?terized by proton nuclear resonance spectroscopy (HNMR). The surface characteristics of Ti?PDA?PEG?PLGA?INH were evaluated by scanning electron microscope (SEM), while drug release behaviors were detected by high performance liquid chromatography (HPLC) and the cumulative release rate was calculated, and carry out the antibacterial performance in vitro. The animal model of femoral condyle bone defect was established in 25 New Zealand white rabbits. Titanium rods covered with PDA?PEG?PLGA?INH coating were implanted into defect area. INH concentrations were detected by HPLC in venous blood, muscle and bone tissue at each time point postoperatively. Another 12 rabbits were randomly divided into experimental group and control group, the experi?mental group was implanted with titanium tablets and titanium rods coated with PDA?PEG?PLGA?INH in the paraspinous muscle and left femoral condyles respectively, while the control group was implanted with a blank sheet of titanium tablets and titanium rods in the same place. Hematoxylin and Eosin Staining were used to observe the biocompatibility of the composite system in vivo at 28 and 56 days postoperatively. Results Ti?PDA?PEG?PLGA?INH controlled drug release coating uniformly distributed on the surface of plates and rods, with translucent form and smooth surface. In vitro INH release kinetics exhibited a short?burst release during the first 8h, and the cumulative release of the INH was about 65%. On the 9th day, the cumulative release of the INH was about 90%, and then the release tended to be flat, and the drug release behavior in vitro continued more than 20d. In vivo release test showed that the concentration of INH in vein blood, muscle and bone tissue around the composite system was increased steadi?ly postoperatively. On about the 28th day, the concentration reached the max. However, the INH concentrations in muscle and bone tissue around the composite system were still higher than the minimum inhibitory concentration (MIC) on the 56th day. The antibacterial test in vitro showed that the titanium tablets coated with PDA?PEG?PLGA?INH formed obvious bacterial inhibition zones. The pathological results indicated that mild inflammatory reaction was seen in the 4th week postoperatively, and the reac?tive capsule formed with loose connective tissue. In the 8th week postoperatively, there's no obvious inflammation occurred, and the reactive capsule became more dense and thicker. Conclusion The study successfully fabricated the Ti?PDA?PEG?PLGA?INH anti?tuberculosis controlled drug release coating, with reasonable release behavior both in vivo and in vitro, effective antibac?terial effect of Mycobacterium tuberculosis in vitro and good tissue biocompatibility, which is a potentially effective drug delivery system for spinal tuberculosis.

Role of focal adhesion kinase in adhesion and migration of Hep G2 cells / 生物医学工程学杂志

Focal adhesion kinase (FAK) plays a critical role in the process of cell adhesion and migration by regulating the expression of downstream small G proteins. A kind of focal adhesion kinase (FAK) inhibitor was used to inhibit the phosphorylation of Y397 site of FAK, and scratch wound migration assay was used to examine the effect of FAK inhibitor with different concentrations (0-250 nmol/mL) on the migration of hepatomal cells (Hep G2 cells) at 0, 2, 4, 8 and 24h. Immunofluorescence analysis and Western blot analysis were performed to detect the expression of F-actin and small G proteins Rac1, RhoA and Cdc42 in Hep G2 cells treated with FAK inhibitor for 120 min. The results indicated that the FAK inhibitor can inhibit the migration of Hep G2 cells with a dose- and time-dependent manner. F-actin was down-regulated in Hep G2 cells treated with FAK inhibitor for 120 min, and expression of small G proteins were inhibited at different durations. The inhibition of FAK phosphorylation could inhibit cell adhesion and migration by down-regulating small G proteins. These results suggested that FAK inhibitor can inhibit the migration of tumor cells by blocking FAK phosphorylation. This means that FAK inhibitor can block the metastasis of tumor cells to surrounding tissues. It may be a potential application in the prevention and treatment of cancer.

Effects of IL-8 on the tight junction of vascular endothelial cells / 国际生物医学工程杂志

Objective To investigate the influence of IL-8 on the tight junction of vascular endothelial cells.Methods Immunofluorescence was used to observe the modality and the distribution of three tight junction proteins (occludin,claudin-5 and ZO-1) of the EA.hy926 cells treated with IL-8 under different concentrations and different times.RT-PCR was used to measure the mRNA expression of these three proteins.Results The results demonstrated that IL-8 could change the distribution of occludin,claudin-5 and ZO-1 in EA.hy926 cells,and the mRNA expression of occludin,claudin-5 and ZO-1 decreased with the increase of IL-8 concentration and treated time.Conclusion The effects of IL-8 on the distribution and the expression of occludin,claudin-5 and ZO-1 are dose and time-dependent.