Objective:To investigate the interventional effects and mechanisms of spermine and spermine derivative 1 (SD1) on human cytomegalovirus (HCMV) infection by establishing an HCMV-infected human bronchial epithelial (HBE) cell model.Methods:HBE cells were used as the study carrier and divided into four groups: a blank control group without HCMV, a control group with HCMV, a spermine group with HCMV and 2 mmol/L spermine, and an SD1 group with HCMV and 2 mmol/L SD1. Viral titers were measured using the 50% tissue culture infectious dose (TCID50) method. The expression of related proteins and genes was detected by enzyme-linked immunosorbent assay (ELISA), Western-blot, and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and cellular reactive oxygen species (ROS) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The above data were compared in multiple groups using one-way analysis of variance, and further pairwise comparisons were conducted using SNK- q test. Results:Compared with the blank control group, the control, spermine, and SD1 groups showed increased viral titers [(0.00±0.00) vs. (8.38±0.50), (6.75±0.46), (5.63±0.52)-log10 TCID50/100 μl; q=21.85, 15.87, and 11.06; all P<0.05]. Compared with the control group, the spermine and SD1 groups exhibited reduced viral titers ( q=5.98 and 10.79; both P<0.05). The SD1 group showed a further reduction compared with the spermine group ( q=4.82, P<0.05). ELISA and RT-qPCR results demonstrated that compared with the blank control group, the control, spermine, and SD1 groups exhibited elevated levels of cyclic guanosine monophosphate-adenosine mompho-sphate (cGAMP), interferon-β (IFN-β), IFN-β mRNA, myxovirus resistance gene 1 (MX1) mRNA, and interferon-stimulated gene 15 (ISG15) mRNA [cGAMP: (0.20±0.03) vs. (0.92±0.10), (1.45±0.09), (2.03±0.15) pmol/ml; IFN-β: (0.13±0.02) vs. (2.34±0.12), (3.50±0.10), (4.50±0.14) ng/ml; IFN-β mRNA: 10.26±1.53 vs. 403.10±15.01, 602.35±11.02, 778.67±24.13; MX1 mRNA: 1.02±0.06 vs. 198.33±7.41, 304.61±9.98, 401.25±10.53; ISG15 mRNA: 1.04±0.08 vs. 273.84±13.71, 396.35±15.20, 489.57±17.46; q=6.93, 12.02, 17.60; 18.43, 28.10, 36.43; 12.52, 19.02, 24.66; 14.30, 21.36, 28.28; 15.12, 20.12, and 25.02; all P<0.05]. Compared with the control group, the spermine and SD1 groups showed further increases in these markers ( q=5.10, 10.68; 9.67, 18.00; 6.50, 12.14; 7.06, 14.00; 5.00, and 9.90; all P<0.05). The SD1 group exhibited even higher expression than the spermine group ( q=5.58, 8.33, 5.64, 6.92, and 4.90; all P<0.05). Western-blot results revealed that compared with the blank control group, the control, spermine, and SD1 groups showed increased expression of phosphorylated TANK-binding kinase 1 (p-TBK1) and phosphorylated interferon regulatory factor 3 (p-IRF3) (p-TBK1: 0.10±0.01 vs. 0.31±0.02, 1.04±0.04, 1.54±0.04; p-IRF3: 0.24±0.02 vs. 0.37±0.02, 0.58±0.03, 1.09±0.04; q=6.57, 29.41, 45.09; 3.38, 8.72, and 21.88; all P<0.05). Compared with the control group, the spermine and SD1 groups exhibited stronger expression ( q=22.84, 38.52; 5.34, and 18.50; all P<0.05). The SD1 group showed further enhancement compared with the spermine group ( q=15.68 and 13.16; both P<0.05). ROS levels were higher in the control group than in the blank control group (1 180.00±16.33 vs. 2 126.67±71.94; q=16.90, P<0.05). Compared with the control group, the spermine and SD1 groups showed reduced ROS expression (2 126.67±71.94 vs. 1 660.00±45.17, 1 473.23±55.58; q=7.30 and 11.66; both P<0.05). The SD1 group exhibited further reduction compared with the spermine group ( q=4.36, P<0.05). Conclusions:In the HCMV-infected HBE cell model, the addition of spermine and SD1 promoted the activation of the cGAMP synthase and type Ⅰ interferon signaling pathway, thereby enhancing innate immune suppression of HCMV infection. Meanwhile, it scavenged ROS to alleviate oxidative stress-induced cellular damage.

Objective To investigate the effect of Pirfenidone(PFN)on respiratory syncytial virus(RSV)infection-induced damage to bronchial epithelial cells by regulating the high mobility group protein B1(HMGB1)/receptor for advanced glycation end products(RAGE)signaling pathway.Methods Human bronchial epithelial cells(HBE)were divided into Control group(cultured for 24 h under normal conditions),RSV group(inoculated with 4.65×106/mL RSV at 33℃for 2 h);low PFN(L-PFN)group(treated with 0.05 mg/mL PFN for 24 h),moderate PFN(M-PFN)group(treated with 0.10 mg/mL PFN for 24 h),high PFN(H-PFN)group(treated with 0.20 mg/mL PFN for 24 h)and recombinant HMGB1(rHMGB1)group(treated with 1 μg/mL rHMGB1+0.20 mg/mL PFN for 24 h).EdU method was applied to detect the proliferation rate of cells in each group,Hochest33258 staining method was applied to detect apoptosis status of cells in each group,and the migration of cells in each group was evaluated by the scratch experiment.Enzyme linked immunosorbent assay(ELISA)was applied to measure the levels of interferon(IFN)-α,IFN-γ,tumor necrosis factor-α(TNF-α),interleukin(IL)-1α,IL-6 and IL-4 in each group of cells,and Western blot was applied to detect the protein expression of HMGB1,RAGE,B lymphoblastoma-2-associated X protein(Bax),cysteine aspartic protease-3(Caspase-3),and B lymphoblastoma-2(Bcl-2).Results Compared with the RSV group,the cell proliferation rate,scratch closure rate,IL-4 levels,and expression of Bcl-2 in L-,M-,and H-PFN groups increased,while the apoptosis rate,the levels of IFN-α,IFN-γ,TNF-α,IL-1α,IL-6,and the expression of HMGB1,RAGE,Bax,and Caspase-3 reduced(P<0.05);rHMGB1 weakened the effect of H-PFN on the above-mentioned indicators(P<0.05).Conclusion PFN may suppress the apoptosis and inflammatory damage of RSV-infected bronchial epithelial cells by inhibiting the HMGB1/RAGE pathway.Conclusion PFN may suppress the apoptosis and inflammatory damage of RSV-infected bronchial epithelial cells by inhibiting the HMGB1/RAGE pathway.

Objective To investigate the effect of Pirfenidone(PFN)on respiratory syncytial virus(RSV)infection-induced damage to bronchial epithelial cells by regulating the high mobility group protein B1(HMGB1)/receptor for advanced glycation end products(RAGE)signaling pathway.Methods Human bronchial epithelial cells(HBE)were divided into Control group(cultured for 24 h under normal conditions),RSV group(inoculated with 4.65×106/mL RSV at 33℃for 2 h);low PFN(L-PFN)group(treated with 0.05 mg/mL PFN for 24 h),moderate PFN(M-PFN)group(treated with 0.10 mg/mL PFN for 24 h),high PFN(H-PFN)group(treated with 0.20 mg/mL PFN for 24 h)and recombinant HMGB1(rHMGB1)group(treated with 1 μg/mL rHMGB1+0.20 mg/mL PFN for 24 h).EdU method was applied to detect the proliferation rate of cells in each group,Hochest33258 staining method was applied to detect apoptosis status of cells in each group,and the migration of cells in each group was evaluated by the scratch experiment.Enzyme linked immunosorbent assay(ELISA)was applied to measure the levels of interferon(IFN)-α,IFN-γ,tumor necrosis factor-α(TNF-α),interleukin(IL)-1α,IL-6 and IL-4 in each group of cells,and Western blot was applied to detect the protein expression of HMGB1,RAGE,B lymphoblastoma-2-associated X protein(Bax),cysteine aspartic protease-3(Caspase-3),and B lymphoblastoma-2(Bcl-2).Results Compared with the RSV group,the cell proliferation rate,scratch closure rate,IL-4 levels,and expression of Bcl-2 in L-,M-,and H-PFN groups increased,while the apoptosis rate,the levels of IFN-α,IFN-γ,TNF-α,IL-1α,IL-6,and the expression of HMGB1,RAGE,Bax,and Caspase-3 reduced(P<0.05);rHMGB1 weakened the effect of H-PFN on the above-mentioned indicators(P<0.05).Conclusion PFN may suppress the apoptosis and inflammatory damage of RSV-infected bronchial epithelial cells by inhibiting the HMGB1/RAGE pathway.Conclusion PFN may suppress the apoptosis and inflammatory damage of RSV-infected bronchial epithelial cells by inhibiting the HMGB1/RAGE pathway.

Objective:To investigate the interventional effects and mechanisms of spermine and spermine derivative 1 (SD1) on human cytomegalovirus (HCMV) infection by establishing an HCMV-infected human bronchial epithelial (HBE) cell model.Methods:HBE cells were used as the study carrier and divided into four groups: a blank control group without HCMV, a control group with HCMV, a spermine group with HCMV and 2 mmol/L spermine, and an SD1 group with HCMV and 2 mmol/L SD1. Viral titers were measured using the 50% tissue culture infectious dose (TCID50) method. The expression of related proteins and genes was detected by enzyme-linked immunosorbent assay (ELISA), Western-blot, and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and cellular reactive oxygen species (ROS) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The above data were compared in multiple groups using one-way analysis of variance, and further pairwise comparisons were conducted using SNK- q test. Results:Compared with the blank control group, the control, spermine, and SD1 groups showed increased viral titers [(0.00±0.00) vs. (8.38±0.50), (6.75±0.46), (5.63±0.52)-log10 TCID50/100 μl; q=21.85, 15.87, and 11.06; all P<0.05]. Compared with the control group, the spermine and SD1 groups exhibited reduced viral titers ( q=5.98 and 10.79; both P<0.05). The SD1 group showed a further reduction compared with the spermine group ( q=4.82, P<0.05). ELISA and RT-qPCR results demonstrated that compared with the blank control group, the control, spermine, and SD1 groups exhibited elevated levels of cyclic guanosine monophosphate-adenosine mompho-sphate (cGAMP), interferon-β (IFN-β), IFN-β mRNA, myxovirus resistance gene 1 (MX1) mRNA, and interferon-stimulated gene 15 (ISG15) mRNA [cGAMP: (0.20±0.03) vs. (0.92±0.10), (1.45±0.09), (2.03±0.15) pmol/ml; IFN-β: (0.13±0.02) vs. (2.34±0.12), (3.50±0.10), (4.50±0.14) ng/ml; IFN-β mRNA: 10.26±1.53 vs. 403.10±15.01, 602.35±11.02, 778.67±24.13; MX1 mRNA: 1.02±0.06 vs. 198.33±7.41, 304.61±9.98, 401.25±10.53; ISG15 mRNA: 1.04±0.08 vs. 273.84±13.71, 396.35±15.20, 489.57±17.46; q=6.93, 12.02, 17.60; 18.43, 28.10, 36.43; 12.52, 19.02, 24.66; 14.30, 21.36, 28.28; 15.12, 20.12, and 25.02; all P<0.05]. Compared with the control group, the spermine and SD1 groups showed further increases in these markers ( q=5.10, 10.68; 9.67, 18.00; 6.50, 12.14; 7.06, 14.00; 5.00, and 9.90; all P<0.05). The SD1 group exhibited even higher expression than the spermine group ( q=5.58, 8.33, 5.64, 6.92, and 4.90; all P<0.05). Western-blot results revealed that compared with the blank control group, the control, spermine, and SD1 groups showed increased expression of phosphorylated TANK-binding kinase 1 (p-TBK1) and phosphorylated interferon regulatory factor 3 (p-IRF3) (p-TBK1: 0.10±0.01 vs. 0.31±0.02, 1.04±0.04, 1.54±0.04; p-IRF3: 0.24±0.02 vs. 0.37±0.02, 0.58±0.03, 1.09±0.04; q=6.57, 29.41, 45.09; 3.38, 8.72, and 21.88; all P<0.05). Compared with the control group, the spermine and SD1 groups exhibited stronger expression ( q=22.84, 38.52; 5.34, and 18.50; all P<0.05). The SD1 group showed further enhancement compared with the spermine group ( q=15.68 and 13.16; both P<0.05). ROS levels were higher in the control group than in the blank control group (1 180.00±16.33 vs. 2 126.67±71.94; q=16.90, P<0.05). Compared with the control group, the spermine and SD1 groups showed reduced ROS expression (2 126.67±71.94 vs. 1 660.00±45.17, 1 473.23±55.58; q=7.30 and 11.66; both P<0.05). The SD1 group exhibited further reduction compared with the spermine group ( q=4.36, P<0.05). Conclusions:In the HCMV-infected HBE cell model, the addition of spermine and SD1 promoted the activation of the cGAMP synthase and type Ⅰ interferon signaling pathway, thereby enhancing innate immune suppression of HCMV infection. Meanwhile, it scavenged ROS to alleviate oxidative stress-induced cellular damage.

Objective:To investigate the value of Breast Ultrasound Report and Data System (BI-RADS) classification in diagnosis of special types of breast cancer.Methods:A total of 112 patients with special type of breast cancer (112 breast lesions) confirmed by pathology were analyzed by using BI-RADS ultrasound category in the Second Affiliated Hospital, Zhejiang University School of Medicine from August 2009 to August 2020. All patients underwent ultrasound before surgery. The breast lesions were evaluated by senior attending and junior resident according to BI-RADS ultrasound category respectively. Taking histopathological result as the gold standard, the sensitivity and accuracy of BI-RADS classification in the diagnosis of special types of breast cancer were calculated.The differences between different special types of breast cancer in terms of ultrasound characteristics and pathological features were analyzed. Kappa consistency test was used to evaluated the consistency of the results of two physicians.Results:In the 112 patients, pathological results showed that there were 20 cases of metaplastic carcinoma, 19 cases of invasive carcinoma with medullary features, 16 cases of differentiated carcinoma of apocrine gland, 12 cases of mucinous carcinoma, 12 cases of invasive micropapillary carcinoma, 10 cases of invasive papillary carcinoma, 6 cases of invasive lobular carcinoma and 17 cases of other special types of carcinoma. Among them, 4 cases (3.5%) were BI-RADS 3, 13 cases (11.6%) were BI-RADS 4a, 42 cases (37.5%) were BI-RADS 4b, 47 cases (42.0%) were BI-RADS 4c and 6 cases (5.4%) were BI-RADS 5. The accuracy and sensitivity of BI-RADS classification in diagnosis of special types of breast cancer was 96.43% and 96.43%, respectively. There was significant difference in BI-RADS grade among different special types of breast cancer ( P<0.05). Most lesions were characterized by hypoechoic with irregular shape and angular or microlobulated margin. The nodule size, boundary, echo and posterior echo in breast cancer with different special types showed significant differences (all P<0.05). There was a good consistency between the two physicians (Kappa=0.789). Conclusions:The ultrasonography features of different special types of breast cancer are different. BI-RADS classification has great value in diagnosis of special types of breast cancer.

Objective:To evaluate the relationships among contrast-enhanced ultrasound (CEUS) features, molecular type, and biomarker expression of breast cancer.Methods:A retrospectively analysis of breast cancer patients confirmed by pathology were performed using Breast Imaging Report And Data System (BI-RADS) ultrasound category lesions in the Second Affiliated Hospital Zhejiang University School of Medicine from May 2020 to April 2021. All patients underwent conventional ultrasound and CEUS before biopsy and/or surgery. The relationships among BI-RADS category, quantitative and qualitative CEUS features and biomarker expression of breast cancer were evaluated.Results:All 149 patients with 149 breast lesions were included. The numbers of BI-RADS category 4A, 4B, 4C, and 5 were 8, 60, 49, and 32, respectively. Among them, the numbers of Luminal A like, Luminal B like (human epidermal growth factor receptor-2 (HER-2) positive), Luminal B like (HER-2 negative), HER-2 overexpression and triple negative type were 81, 29, 17, 15, and 7. No significant correlations were found among BI-RADS category, molecular types, and biomarker estrogen receptor (ER), progesterone receptor (PR), HER-2, and antigen Ki-67 (Ki-67) expression (all P>0.05). There were no correlations between quantitative or qualitative CEUS features and molecular types of breast cancer (all P>0.05). There were no correlations between qualitative CEUS variables and ER, PR, HER-2, and Ki-67 expression (all P>0.05). Ascending slope (AS) were negatively correlated with ER and PR expression( r=-0.40, P=0.01; r=-0.35, P=0.03). Descending slope (DS) were positively correlated with ER and PR expression( r=0.42, P=0.01; r=0.36, P=0.03). Arrive time (AT) were positively correlated with HER-2 expression( r=0.37, P=0.02). Conclusions:AS and DS are correlated with ER and PR expression.Arrive time (AT) is correlated with HER-2 expression. The quantitative variables of CEUS are helpful for evaluation of biomarker expression in breast cancer.

Background and purpose:Triple negative breast cancer (TNBC) is with high invasion, poor prognosis and lack of usefull treatment. This study investigated expression status of ICAM-1 protein in TNBC in order to explore its relationship with clinicopathological features and outcome in patients.Methods:Fifty-nine tissue samples of TNBC were collected while 50 cases of para-carcinoma tissue samples were used as negative controls. Immunohistochemical staining was conducted to detect expression level of ICAM-1 protein. The relationship of ICAM-1 protein expression with clinicopathological features (age, tumor size, subtype, grade, status of lymph node metastasis, TNM stage, vascular tumor thrombus, nerve inifltration, Ki-67, p53 and E-cadherin expression) and outcome in patients were analyzed.Results:The ICAM-1 protein expression of TNBC was signiifcantly higher than that in adjacent tissues (P=0.000). ICAM-1 expression was related to status of lymph node metastasis, grade and TNM stage (with aP-value of 0.036, 0.027 and 0.048, respectively), while demonstrated an undeifned relationship with tumor size, subtype, vascular tumor thrombus and expression of Ki-67, p53 and E-cadherin. The disease-free survival (DFS) of ICAM-1 high expression set was shorter than that of the lower one but has nothing to do with overall survival (OS). In addition, Cox proportional hazards model showed that ICAM-1 expression and lymph node metastasis were independent risk factors of DFS in patients (HR=3.2, 95%CI: 1.6 to 6.4, HR=2.7, 95%CI: 1.28 to 5.9,P<0.05).Conclusion:ICAM-1 could serve as a predictive factor for differentiation status of TNBC. The high expression of ICAM-1 in TNBC may indicate poorer prognosis.

Background and purpose:The time from ifrst onset of symptoms or signs to a deifnitive diagnosis is deifned as diagnostic interval (DI). The relation of DI to other clinicopathological parameters andthe impact of DI on prognosis of patients with triple-negative breast cancer (TNBC) remain unclear.This article plans to make an intensive study of these questions.Methods:The clinical records of a series of 83 consecutively presenting unselected patients referred to the Shanghai Sixth People’s Hospital with diagnosed TNBC between September 2009 and September 2015 were retrospectively reviewed. Clinical and pathological factors included were investigated by univariate analysis using the Kaplan-Meier method, the factors associated with prognosis were further evaluated by multivariable analysis with Cox progression model.t-test and Kruskal-Wallis test were used to study the correlation between DI and other characters.Results:DI: stage T3>T1 (P=0.01), stageⅢ>Ⅱ (P=0.03) andⅠ (P=0.01). Compared with patients of DI≥3 months, the <3 months group had earlier age (P=0.028) and TNM stage (P=0.035). T stage, N stage, neoadjuvant chemotherapy, TNM stage and DI are inlfuencing factors of overall survival (OS). Age, T stage, N stage, TNM stage, menstrual status and neoadjuvant chemotherapy are inlfuencing factors of progression-free survival (PFS). TNM staging is an independent inlfuencing factor for OS and PFS.Conclusion:Patients with later disease stage were more likely to have a longer DI; The shorter DI, the earlier age and stage of disease; DI is the inlfuence factor of OS; TNM stage is an independent inlfuencing factor for OS and PFS.

Aim To observe the effect of ZDY102, a C25 stereo-isomer of ZMS, the active component of Zhimu, on brain M receptor density of dementia model animals and the correlation with its effect on learning/memory ability. Methods The rats were randomly divided into five groups: control group, model group, model given orally for 2 months with 3.6 mg?kg -1?d -1 of ZDY102 treatment, model treated with 9.0 mg?kg -1?d -1 of ZDY102, and model treated with 18.0 mg?kg -1?d -1 of ZDY102. Dementia model was produced by single unilateral injection of 4 ?l of normal saline containing 4 ?g of ?-amyloid (25～35) and 1 ?g of ibotenic acid into right basal ganglion region with the aid of a stereotaxic equipment. The brain muscarinic receptor density was analyzed with single-site binding assay using 3H-quinuclidinyl benzilae(QNB). The learning/memory ability was measured by Y-maze performance. Results Two months after model production, the learning and memory ability as well as the density of muscarinic receptor in brain were significantly decreased in model rats compared with those in control rats. Parallel models treated with daily oral administration of ZDY102 for two months improved in learning and memory ability and their muscarinic receptor density was significantly increased when compared with model rats. The correlation coefficient between total M receptor densities and the learning/memory ability was significant when examined with linear regression. Conclusion ZDY102 can significantly improve the learning and memory ability and increase the brain muscarinic receptor density of the model. Since brain muscarinic receptors are closely correlated to learning and memory, up-regulation of M receptor density might play a very important role in the therapeutic effect of ZDY102.