Objective The purpose of this study was to provide a more effective method for researching the prevention and treatment of Graves'disease by comparing the effects of two plasmid vectors expressing the human thyrotropin receptor(TSHR)A subunit gene in inducing an animal model of Graves'disease via electroporation.Methods Plasmids pcDNA3.1-THSR A,and pTriEx1.1-THSR A expressing the TSHR A subunit were constructed and used to induce Graves'disease by intramuscular injection with immediate electroporation once every 3 weeks for a total of 4 times.Mice in the control group were injected with PBS.One week after the second electroporation,blood was collected to measure serum thyrotropin receptor antibody(TRAb).Three weeks following the last electroporation,echocardiography was performed on the mice.Mice were sacrificed 4 weeks after the last electroporation;blood,thyroid,and orbital tissues were collected;serum total thyroxine(TT4)was measured;and histological examination was performed.Results The average concentrations of serum TRAb in the pcDNA3.1-TSHR A group(n=15)and the pTriEx1.1-TSHR A group(n=13)were(6.9±2.0)U/L and(7.5±2.2)U/L,respectively.The latter was significantly higher than that in the control group(4.9±0.5)U/L(P=0.033).The average concentrations of serum TT4 in the pcDNA3.1-TSHR A group and pTriEx1.1-TSHR A group were(41.4±23.8)ng/mL and(63.2±53.7)ng/mL,respectively,both higher than that in the control group:(20.2±4.0)ng/mL(P＜0.01).Thyroid pathology showed thyroid follicular epithelial hyperplasia with T-cell infiltration in the model group.Echocardiography showed that the left ventricle mass in the pTriEx1.1-TSHR A group was higher than those in the control group(P=0.007)and pcDNA3.1-TSHR A group(P=0.012).Orbital pathology showed fibrotic changes in the extraocular muscles of mice in the model groups.Conclusions Both pcDNA3.1 and pTriEx1.1 expressing the TSHR A subunit were able to induce Graves'disease in mice by electroporation,and the efficiency of the two plasmids in inducing hyperthyroidism and Graves'ophthalmopathy was similar.The efficiency of pTriEx 1.1-TSHR A in inducing thyrotoxic heart disease was better than that of pcDNA3.1-TSHR A.

Objective:To investigate the effects of lipopolysaccharide (LPS)-activated stimulator of interferon genes (STING) signaling on the biological behavior of periodontal ligament cells and its mechanism of action.Methods:Human periodontal ligament cells (hPDLC) were divided into the PBS group and the LPS group by stimulated with phosphate-buffered saline (PBS) and LPS derived from Porphyromonas gingivalis (ATCC 33277) for 12 hours, respectively. The intracellular distribution of 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA damage, and the activation level of STING signaling were detected by immunofluorescence. The source of intracellular double-stranded DNA was detected by live-cell probes. The levels of osteogenic-related proteins, such as special protein 7 (SP7), bone morphogenetic protein 2 (BMP2), cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), and STING were detected by Western blotting. The cell supernatants of the PBS group and the LPS group were collected, and the levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and interferon (IFN)-β, were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). A total of 12 cgas knockout mice and 12 littermate wild-type mice were constructed. The maxillary second molars of the mice were ligated with silk or sham surgery, respectively. After 7 days of modeling, the mice were divided into littermate control sham surgery group, littermate control periodontitis group, cgas knockout sham surgery group, and cgas knockout periodontitis group, with 6 mice in each group. Micro-CT was used to collect image data, and three-dimensional reconstructions were performed on the maxillary samples of each group. The distance from the cemento-enamel junction to the alveolar bone crest (CEJ-ABC), bone volume fraction (BV/TV), and bone mineral density (BMD) in the model area were statistically analyzed using CTAn and CTVOX software. Frozen sectioning was used to obtain sections of the maxillary molars of each group of mice, and the signal intensities of cGAS and STING proteins were detected by immunofluorescence. Results:Immunofluorescence results showed that the fluorescence signal intensity of 8-OHdG outside the nucleus in the LPS group (4.09±0.24) was significantly higher than that in the PBS group (1.00±0.10) ( t=20.33, P<0.001). The co-localization signal of mitochondrial marker TOM20 and 8-OHdG (8.56±0.53) were significantly higher than that of PBS group (1.00±0.09) ( t=24.37, P<0.001). Live cell DNA probe detection showed that the signal intensity of double-stranded DNA in LPS group (3.23±0.12) was significantly stronger than that in PBS group (1.00±0.17) ( t=18.30, P<0.001). Immunofluorescence demonstrated a significant increase in STING expression in hPDLC of the LPS group ( t=6.42, P<0.001), and it was colocalized with the Golgi marker GM130. ELISA results showed that the abundance of IL-6, IFN-β, and IL-1β in the supernatant of the LPS group were higher than those of the PBS group ( t=12.44, t=11.38, t=9.48, all P<0.001). Animal experiments confirmed that compared with the sham operation group [(207.61±38.09) and (238.97±45.90) μm], the CEJ-ABC in the periodontitis group [(420.31±35.32) and (405.16±35.51) μm] were increased ( P<0.01), while the CEJ-ABC in the cgas knockout periodontitis group [(295.11±35.43) and (309.15±32.22) μm] were significantly lower than those in the control periodontitis group of the same litter ( P<0.01). Compared with the sham operation group (45.84±6.41), the STING fluorescence signal in the periodontitis group (152.44±6.86) was significantly increased ( P<0.001). Compared with the control periodontitis group of the same litter, the STING signal in the cgas knockout periodontitis group was significantly reduced (88.31±9.70) ( P<0.001). Conclusions:LPS stimulation can activate the STING signal by generating mitochondrial-derived double-stranded DNA, stimulating hPDLC to secrete inflammatory cytokines and impairing osteogenic differentiation potential. Suppressing STING activation in animal models can reduce bone destruction in periodontitis.

Objective:To investigate the effects of lipopolysaccharide (LPS)-activated stimulator of interferon genes (STING) signaling on the biological behavior of periodontal ligament cells and its mechanism of action.Methods:Human periodontal ligament cells (hPDLC) were divided into the PBS group and the LPS group by stimulated with phosphate-buffered saline (PBS) and LPS derived from Porphyromonas gingivalis (ATCC 33277) for 12 hours, respectively. The intracellular distribution of 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA damage, and the activation level of STING signaling were detected by immunofluorescence. The source of intracellular double-stranded DNA was detected by live-cell probes. The levels of osteogenic-related proteins, such as special protein 7 (SP7), bone morphogenetic protein 2 (BMP2), cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), and STING were detected by Western blotting. The cell supernatants of the PBS group and the LPS group were collected, and the levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and interferon (IFN)-β, were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). A total of 12 cgas knockout mice and 12 littermate wild-type mice were constructed. The maxillary second molars of the mice were ligated with silk or sham surgery, respectively. After 7 days of modeling, the mice were divided into littermate control sham surgery group, littermate control periodontitis group, cgas knockout sham surgery group, and cgas knockout periodontitis group, with 6 mice in each group. Micro-CT was used to collect image data, and three-dimensional reconstructions were performed on the maxillary samples of each group. The distance from the cemento-enamel junction to the alveolar bone crest (CEJ-ABC), bone volume fraction (BV/TV), and bone mineral density (BMD) in the model area were statistically analyzed using CTAn and CTVOX software. Frozen sectioning was used to obtain sections of the maxillary molars of each group of mice, and the signal intensities of cGAS and STING proteins were detected by immunofluorescence. Results:Immunofluorescence results showed that the fluorescence signal intensity of 8-OHdG outside the nucleus in the LPS group (4.09±0.24) was significantly higher than that in the PBS group (1.00±0.10) ( t=20.33, P<0.001). The co-localization signal of mitochondrial marker TOM20 and 8-OHdG (8.56±0.53) were significantly higher than that of PBS group (1.00±0.09) ( t=24.37, P<0.001). Live cell DNA probe detection showed that the signal intensity of double-stranded DNA in LPS group (3.23±0.12) was significantly stronger than that in PBS group (1.00±0.17) ( t=18.30, P<0.001). Immunofluorescence demonstrated a significant increase in STING expression in hPDLC of the LPS group ( t=6.42, P<0.001), and it was colocalized with the Golgi marker GM130. ELISA results showed that the abundance of IL-6, IFN-β, and IL-1β in the supernatant of the LPS group were higher than those of the PBS group ( t=12.44, t=11.38, t=9.48, all P<0.001). Animal experiments confirmed that compared with the sham operation group [(207.61±38.09) and (238.97±45.90) μm], the CEJ-ABC in the periodontitis group [(420.31±35.32) and (405.16±35.51) μm] were increased ( P<0.01), while the CEJ-ABC in the cgas knockout periodontitis group [(295.11±35.43) and (309.15±32.22) μm] were significantly lower than those in the control periodontitis group of the same litter ( P<0.01). Compared with the sham operation group (45.84±6.41), the STING fluorescence signal in the periodontitis group (152.44±6.86) was significantly increased ( P<0.001). Compared with the control periodontitis group of the same litter, the STING signal in the cgas knockout periodontitis group was significantly reduced (88.31±9.70) ( P<0.001). Conclusions:LPS stimulation can activate the STING signal by generating mitochondrial-derived double-stranded DNA, stimulating hPDLC to secrete inflammatory cytokines and impairing osteogenic differentiation potential. Suppressing STING activation in animal models can reduce bone destruction in periodontitis.

Objective The purpose of this study was to provide a more effective method for researching the prevention and treatment of Graves'disease by comparing the effects of two plasmid vectors expressing the human thyrotropin receptor(TSHR)A subunit gene in inducing an animal model of Graves'disease via electroporation.Methods Plasmids pcDNA3.1-THSR A,and pTriEx1.1-THSR A expressing the TSHR A subunit were constructed and used to induce Graves'disease by intramuscular injection with immediate electroporation once every 3 weeks for a total of 4 times.Mice in the control group were injected with PBS.One week after the second electroporation,blood was collected to measure serum thyrotropin receptor antibody(TRAb).Three weeks following the last electroporation,echocardiography was performed on the mice.Mice were sacrificed 4 weeks after the last electroporation;blood,thyroid,and orbital tissues were collected;serum total thyroxine(TT4)was measured;and histological examination was performed.Results The average concentrations of serum TRAb in the pcDNA3.1-TSHR A group(n=15)and the pTriEx1.1-TSHR A group(n=13)were(6.9±2.0)U/L and(7.5±2.2)U/L,respectively.The latter was significantly higher than that in the control group(4.9±0.5)U/L(P=0.033).The average concentrations of serum TT4 in the pcDNA3.1-TSHR A group and pTriEx1.1-TSHR A group were(41.4±23.8)ng/mL and(63.2±53.7)ng/mL,respectively,both higher than that in the control group:(20.2±4.0)ng/mL(P＜0.01).Thyroid pathology showed thyroid follicular epithelial hyperplasia with T-cell infiltration in the model group.Echocardiography showed that the left ventricle mass in the pTriEx1.1-TSHR A group was higher than those in the control group(P=0.007)and pcDNA3.1-TSHR A group(P=0.012).Orbital pathology showed fibrotic changes in the extraocular muscles of mice in the model groups.Conclusions Both pcDNA3.1 and pTriEx1.1 expressing the TSHR A subunit were able to induce Graves'disease in mice by electroporation,and the efficiency of the two plasmids in inducing hyperthyroidism and Graves'ophthalmopathy was similar.The efficiency of pTriEx 1.1-TSHR A in inducing thyrotoxic heart disease was better than that of pcDNA3.1-TSHR A.

ObjectiveTo investigate the correlation between mild cognitive impairment (MCI) and traditional Chinese medicine (TCM) body constitution types in the elderly, and to provide an evidence for the control of cognitive impairment in the elderly. MethodsThe elderly aged 65 and above who participated in the community physical examinations in a community of Changning District, Shanghai were selected as the research subjects. The cognitive function was assessed by using the Clock Drawing Test combined with the Ascertain Dementia 8-item Questionnaire (AD8), the Montreal Cognitive Assessment (MoCA) and the Mini-mental State Examination (MMSE). The diagnostic criteria for MCI was identified based on the 2018 Chinese Guidelines for the Diagnosis and Treatment of Dementia and Cognitive Disorders, along with the assessment results and clinical history information. The current investigation method was used to collect the basic information and the prevalence of chronic disease of the subjects through questionnaire inquiries. The elderly subjects’ ability to take care of themselves was evaluated based on the Elderly Self⁃Care Ability Evaluation Scale, while the TCM body constitution types were determined based on the Chinese Medicine Health Care Management Service Specification. The association of the detection rate of MCI with gender, education level, history of chronic disease and TCM body constitution types were analyzed lastly. ResultsA total of 2 351 elderly people were investigated, including 1 037 males and 1 314 females, with an average age of (74.11±6.15) years. 174 subjects, accounting for 7.40%, were identified with MCI. The highest detection rate of MCI in the elderly are those with a Qi stagnation constitution (10.8%), followed by those with a dampness-heat constitution (9.1%) and a Qi deficiency constitution (8.4%). Multivariate logistic regression analysis showed that advanced age, lower educational level, a history of tuberculosis, and TCM constitutions such as dampness-heat, Qi stagnation, and Qi deficiency were the potential risk factors for MCI. ConclusionThere is a significant association between TCM constitution types such as dampness-heat, Qi stagnation, and Qi deficiency with MCI. TCM techniques can be integrated into the health management services for the elderly population, and targeted interventions can be provided to those with imbalanced constitution types so as to reduce the risk of MCI.

Objective:To evaluate the effectiveness of deep learning reconstruction (DLR) compared with hybrid iterative reconstruction (Hybrid IR) in improving the image quality in chest low-dose CT (LDCT).Methods:Seventy-seven patients who underwent LDCT scan for physical examination or regular follow-up in Peking Union Medical College Hospital from October 2020 to March 2021 were retrospectively included. The LDCT images were reconstructed with Hybrid IR at standard level (Hybrid IR Stand) and DLR at standard and strong level (DLR Stand and DLR Strong). Regions of interest were placed on pulmonary lobe, aorta, subscapularis muscle and axillary fat to measure the CT value and image noise. The signal to noise ratio (SNR) and contrast to noise ratio (CNR) were calculated. Subjective image quality was evaluated using Likert 5-score method by two experienced radiologists. The number and features of ground-glass nodule (GGN) were also assessed. If the scores of the two radiologists were inconsistent, the score was determined by the third radiologist. The objective and subjective image evaluation were compared using the Kruskal-Wallis test, and the Bonferroni test was used for multiple comparisons within the group.Results:Among Hybrid IR Stand, DLR Stand and DLR Strong images, the CT value of pulmonary lobe, aorta, subscapularis muscle and axillary fat had no significant differences (all P>0.05), but the image noise and SNR of pulmonary lobe, aorta, subscapularis muscle and axillary fat had significant differences(all P<0.05), and the CNR of images had significant difference( P<0.05), too. The CNR of Hybrid IR Stand images, DLR stand images and DLR strong images were 0.71 (0.49, 0.88), 1.06 (0.78, 1.32) and 1.14 (0.84, 1.48), respectively. Compared with Hybrid IR images, DLR images had lower objective and subjective image noise,higher SNR and CNR (all P<0.05). The scores of DLR images were superior to Hybrid IR images in identifying lung fissures, pulmonary vessels, trachea and bronchi, lymph nodes, pleura, pericardium and GGN (all P<0.05). Conclusions:DLR significantly reduced the image noise, and DLR images were superior to Hybrid IR images in identifying GGN in chest LDCT while maintaining superior image quality at relatively low radiation dose levels. Thus DLR images can improve the safety of lung cancer screening and pulmonary nodule follow-up by CT.

Objective:To explore the effect of deep learning reconstruction (DLR) on radiation dosage reduction and image quality of CTPA compared with hybrid iterative reconstruction (HIR).Methods:A total of 100 patients with suspected pulmonary embolism (APE) or indications for CTPA due to other pulmonary artery diseases in Peking Union Medical College Hospital from December 2020 to April 2021 were prospectively enrolled and divided into HIR group and DLR group according to block randomization, with 50 cases in each group. The patient′s gender, age and body mass index (BMI) were recorded. HIR group and DLR group underwent standard deviation (SD)=8.8 and SD=15 CTPA protocols in combination with HIR and DLR algorithm respectively. Other scanning parameters and contrast medium injection plan were the same. The effective dose (ED) and size-specific dose estimate (SSDE) were calculated. Regions of interest (ROIs) were drawn in the lumen of Grade 1-3 pulmonary arteries and bilateral paravertebral muscles. The corresponding CT and SD values were recorded to acquire signal to noise ratio (SNR) and contrast noise ratio (CNR). Based on a double-blind method, two radiologists evaluated the subjective noise, visualization of pulmonary arteries, and diagnostic confidence of the two groups by 5-point Likert scales. The inconsistent results were judged comprehensively by the third radiologist. Independent samples t-test was used to compare the demographic data, radiation dosage and quantitative image quality of the two groups. Mann-Whitney U test was used to compare the subjective noise, visualization of pulmonary arteries and diagnostic confidence between the two groups. Linear weighted Kappa coefficient was calculated to analyze the consistency of the qualitative scores between the two radiologists. Results:There were no significant differences in gender, age and BMI between the two groups ( P>0.05). The CT values of Grade1-3 pulmonary arteries and paravertebral muscle had no significant differences ( P>0.05). Compared with HIR group, the ED and SSDE in DLR group decreased by about 35% to 1.3 mSv and 4.20 mGy respectively, while the SNR (30±5) and CNR (26±5) of CTPA images were higher in DLR group than those in HIR group (23±5 and 20±5, with t=-6.60 and -5.90, respectively, both P<0.001). The subjective noise score was higher in DLR group than that in HIR group ( Z=-7.34, P<0.001). In addition, two radiologists showed excellent interobserver agreement in DLR group (Kappa=0.847, 95%CI 0.553-1.000). No significant differences were found in visualization of pulmonary arteries and diagnostic confidence between the two groups ( P>0.05). Conclusion:DLR further reduced the radiation dosage and improved the image quality of CTPA, with no detriment to diagnostic confidence. Thus DLR is worthy of clinical promotion.

Objective:To investigate the feasibility of chest ultra-low dose CT (ULDCT) using deep learning reconstruction (DLR) for lung cancer screening, and to compare its image quality and nodule detection rate with ULDCT iterative reconstruction (Hybrid IR) and conventional dose CT (RDCT) Hybrid IR.Methods:The patients who underwent chest CT examination for pulmonary nodules in Peking Union Medical College Hospital from October 2020 to March 2021 were prospectively included and underwent chest RDCT (120 kVp, automatic tube current), followed by ULDCT (100 kVp, 20 mA). The RDCT images were reconstructed with Hybrid IR (adaptive iterative dose reduction 3D,AIDR 3D), and ULDCT was reconstructed with AIDR3D and DLR. Radiation dose parameters and nodule numbers were recorded. Image quality was assessed using objective noise, signal-to-noise ratio (SNR) of the main trachea and left upper lobe, subjective image scores of the lung and nodules. Subjective scores were scored by 2 experienced radiologists on a Likert 5-point scale. The difference of radiation dose was compared with paired t-test between ULDCT and RDCT.The differences of quantitative indexes, objective image noise and subjective scores of the three reconstruction methods were compared with one-way analysis of variance or Friedman test. Results:Forty-five patients were enrolled, including 17 males and 28 females, aged from 32 to 74 (55±11) years. The radiation dose of ULDCT was (0.17±0.01) mSv, which was significantly lower than that of RDCT [(1.35±0.41) mSv, t=15.46, P<0.001]. There were significant differences in the image noise and SNR in the trachea and lung parenchyma and in the CT value of the trachea among ULDCT-AICE, ULDCT-AIDR 3D and RDCT-AIDR 3D images ( P<0.05). Image noise in the trachea and lung parenchyma and CT value in the trachea of ULDCT-AICE were significantly lower than those of ULDCT-AIDR 3D ( P<0.05) and comparable to RDCT-AIDR 3D ( P>0.05). There were significant differences in subjective image scores of the lung and nodules among ULDCT-AICE, ULDCT-AIDR 3D and RDCT-AIDR 3D images (χ2=50.57,117.20, P<0.001). Subjective image scores of the lung and nodules for ULDCT-AICE were significantly higher than those of ULDCT-AIDR 3D ( P<0.05), and non-inferior to RDCT-ADIR 3D ( P>0.05). All 72 clinically significant nodules detected on RDCT-ADIR 3D were also noted on ULDCT-AICE and ULDCT-AIDR 3D images. Conclusions:Chest ULDCT using DLR can significantly reduce the radiation dose, and compared with Hybrid IR, it can effectively reduce the image noise and improve SNR, and display the pulmonary nodules well. The image quality and nodule detection are not inferior to RDCT Hybrid IR routinely used in clinical practice.

Objective To investigate the effect of quercetin on suppressing the proliferation,migration and invasion of A549 cells via the signal transducer and activator of transcription 3(STAT3)signaling pathway. Methods The A549 cells were cultured in vitro and treated with quercetin at various concentrations(0,7.5,15,30,60 and 120μmol/L)for 24 h,48 h and 72 h. The proliferation of A549 and the 50%inhibitory concentration(IC50)were measured by the cell counting kit-8(CCK-8). The A549 cells treated for 24 h were randomly divided into 4 groups:the blank control,15 and 30 μmol/L quercetin,and 3 μg/ml cisplatin(the positive control) groups. The effect of quercetin on adhesion rate was detected by the cell adhesion assay;the cell migration ability was evaluated by the wound healing assay;the cell invasion ability was evaluated by the Transwell chamber assay;the expression of STAT3 and phosphory?lated-STAT3(p-STAT3)proteins were detected by Western blot assay. Results Quercetin inhibited A549 cell growth dose-depend?ently. Compared with the blank control group,quercetin could significantly inhibit the adhesion rate,migration ability and invasion of A549 cells(P<0.05 or P<0.01);compared with the blank control group,quercetin significantly inhibited STAT3 and p-STAT3 ex?pression level(P<0.05 or P<0.01). Conclusion Quercetin could inhibit the proliferation,migration and invasion of A549 cells, and the mechanism is libely related to the STAT3 signal pathway.

BACKGROUND:The methods used to repair articular cartilage defects currently have the cons and pros. Fibrocartilages are commonly used to repair tissues, and the fibrocartilage lacks of the tissue biomechanical properties and chemical properties of normal hyaline cartilage. OBJECTIVE:To investigate the feasibility of biological osteochondral xenogenic graft transplantation to repair articular cartilage defects. METHODS:The normal goats were randomly divided into two groups. The donor pig knee joints were the experimental group. Cylindrical osteochondral with the diameter of 4.5 mm and length of 10 mm were col ected with the Smith&Nephew osteochondral transplantation device, and the patented technology was used for deantigen. The donor goat knee joint osteochondrals were the control group and preserved with cryopreservation. The lesions on femoral trochlea and weight-bearing surface of medial condyle were selected respectively for osteochondral implantation, and the animals were sacrificed at 16 and 32 weeks after operation for the general and pathological section observation. RESULTS AND CONCLUSION:General observation in the experimental showed that the lesions were covered by fibroid tissue;some cartilage of the grafts turned yel ow and there was clear boundary between the surface and the peripheral cartilages;the general and section observation under microscope showed that lesions of the control group were covered by the grafts basical y, and cracks could be seen on the edge of the transplant part. The results show that there is difference between effects of biological osteochondral xenogenic graft transplantation and osteochondral al ograft transplantation for the repairing of articular cartilage defects, and osteochondral al ograft transplantation bas better effect.