BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

Objectives:To explore whether short-course radiotherapy (SCRT)-based total neoadjuvant therapy (TNT) combined with PD-1 inhibitors could further promote tumor regression and improve the prognosis.Methods:This is a prospective, multicenter, two-arm randomized controlled, seamless phase Ⅱ/Ⅲ trial for proficient mismatch repair or microsatellite stable (pMMR/MSS) locally advanced rectal cancer (LARC). Eligible patients were randomly assigned to the iTNT (TNT+PD-1) group or the TNT group. Patients in the TNT group received SCRT (5 Gy×5) followed by 4 cycles of CAPOX or 6 cycles of mFOLFOX chemotherapy, with the iTNT group receiving SCRT followed by the same regime in combination with 4 cycles of Sintilimab. Total mesorectal excision (TME) surgery or watch and wait (W&W) was performed after neoadjuvant therapy and then 2 cycles of same regimen as before were recommended. The primary endpoints are the complete response (CR) rate for phase Ⅱ trial and 3-year disease-free survival (DFS) for phase Ⅲ trial. A total of 588 patients will be enrolled for the phase Ⅱ/Ⅲ trial. Short-term efficacy and safety data from the initial 100 treated patients were analyzed as planned.Results:From 2022-8-31 to 2023-5-24 the initial 100 patients were enrolled from 10 hospitals in China, 76.0%(76/100) patients were male, and the median age was 61 years (21-74 years). More patients had tumors located in the lower rectum (78.0%, 78/100), staged T3-4 (97.0%, 97/100) and N1-2 (93.0%, 93/100), and about half of the tumors invaded the mesorectal fascia (52.0%, 52/100) and with extramural vascular invasion (51.0%, 51/100). Analyses were performed according to the per-protocal (PP) set. All patients in the iTNT group ( n=52) and the TNT group ( n=48) completed SCRT; The 4-cycle chemotherapy±Sintilimab completion rates were 86.5% and 100.0% in the iTNT and TNT groups, respectively. In the iTNT group, 82.7% (43/52), 11.5% (6/52), and 5.8% (3/52) of the patients received 4, 3, and 2 cycles of PD-1 inhibitor. After TNT, 68 patients underwent radical surgery and 15 patients achieved cCR and adopted W&W. The pathological complete response (pCR) rates were 48.5% (16/33) and 17.1% (6/35) in the iTNT and TNT groups, with CR rates of 50.0% (25/50) and 26.1% (12/46), respectively. The incidence of treatment-related grade 3-4 adverse events was 26.9% (14/52, iTNT group) and 18.8% (9/48, TNT group), with thrombocytopenia and leukopenia being the most common. Among patients receiving immunotherapy, grade 3 immunotherapy-related adverse events occurred in 2 (3.8%, 2/52) patients: one case was pancreatitis, another case was hepatitis combined with myositis and myocarditis. Conclusion:The preliminary results show that SCRT-based TNT combined with PD-1 inhibitors could further improve the CR rate for LARC without unexpected serious adverse events.

Objective:To investigate the changes of upper airway and adenoids in children with hypertrophic adenoid skeletal classⅠ and class Ⅲ anterior crossbite after orthodontic treatment.Methods:From January 2021 to January 2022,155 children with skeletal class Ⅲ anterior crossbite who were treated with anterior traction in Stomatological Hospital,Shanxi Medical University were selected.They were divided into class Ⅲ normal group(72 cases)and class Ⅲ hypertrophic group(83 cases)according to whether the adenoids were hypertrophic.A total of 122 children with class Ⅰ anterior counterocclusion were treated with"2×4"treatment,which were di-vided into Class Ⅰ normal group(60 cases)and class Ⅰ hypertrophy group(62 cases).The changes of upper airway and adenoid were compared between the two groups.Results:(1)After intervention,only the width of nasopharynx cavity increased(P＜0.05)in Class Ⅰ normal group and Class Ⅰ hypertrophic group,and there was no significant difference in other indicators(P＞0.05).(2)After intervention,the adenoid thickness and A/N ratio of children with skeletal class Ⅲ anterior crossbite decreased,and the width of na-sopharynx cavity,nasopharynx airway space,soft palate upper and rear airway space,uvula tip rear airway space,the minimum air-way space between soft palate and adenoid,and mandibular plane angle increased(P＜0.05).The A/N ratio of class Ⅲ hypertrophic group was lower than that of class Ⅲ normal group(P＜0.05).Conclusion:Orthodontic treatment can reduce the degree of adenoid hypertrophy and expand the airway in children with skeletal class Ⅲ anterior crossbite.Only the width of the nasopharynx cavity can be widened after the correction intervention for children with adenoid hypertrophy and skeletal class Ⅰ anterior crossbite.

Objectives:To explore whether short-course radiotherapy (SCRT)-based total neoadjuvant therapy (TNT) combined with PD-1 inhibitors could further promote tumor regression and improve the prognosis.Methods:This is a prospective, multicenter, two-arm randomized controlled, seamless phase Ⅱ/Ⅲ trial for proficient mismatch repair or microsatellite stable (pMMR/MSS) locally advanced rectal cancer (LARC). Eligible patients were randomly assigned to the iTNT (TNT+PD-1) group or the TNT group. Patients in the TNT group received SCRT (5 Gy×5) followed by 4 cycles of CAPOX or 6 cycles of mFOLFOX chemotherapy, with the iTNT group receiving SCRT followed by the same regime in combination with 4 cycles of Sintilimab. Total mesorectal excision (TME) surgery or watch and wait (W&W) was performed after neoadjuvant therapy and then 2 cycles of same regimen as before were recommended. The primary endpoints are the complete response (CR) rate for phase Ⅱ trial and 3-year disease-free survival (DFS) for phase Ⅲ trial. A total of 588 patients will be enrolled for the phase Ⅱ/Ⅲ trial. Short-term efficacy and safety data from the initial 100 treated patients were analyzed as planned.Results:From 2022-8-31 to 2023-5-24 the initial 100 patients were enrolled from 10 hospitals in China, 76.0%(76/100) patients were male, and the median age was 61 years (21-74 years). More patients had tumors located in the lower rectum (78.0%, 78/100), staged T3-4 (97.0%, 97/100) and N1-2 (93.0%, 93/100), and about half of the tumors invaded the mesorectal fascia (52.0%, 52/100) and with extramural vascular invasion (51.0%, 51/100). Analyses were performed according to the per-protocal (PP) set. All patients in the iTNT group ( n=52) and the TNT group ( n=48) completed SCRT; The 4-cycle chemotherapy±Sintilimab completion rates were 86.5% and 100.0% in the iTNT and TNT groups, respectively. In the iTNT group, 82.7% (43/52), 11.5% (6/52), and 5.8% (3/52) of the patients received 4, 3, and 2 cycles of PD-1 inhibitor. After TNT, 68 patients underwent radical surgery and 15 patients achieved cCR and adopted W&W. The pathological complete response (pCR) rates were 48.5% (16/33) and 17.1% (6/35) in the iTNT and TNT groups, with CR rates of 50.0% (25/50) and 26.1% (12/46), respectively. The incidence of treatment-related grade 3-4 adverse events was 26.9% (14/52, iTNT group) and 18.8% (9/48, TNT group), with thrombocytopenia and leukopenia being the most common. Among patients receiving immunotherapy, grade 3 immunotherapy-related adverse events occurred in 2 (3.8%, 2/52) patients: one case was pancreatitis, another case was hepatitis combined with myositis and myocarditis. Conclusion:The preliminary results show that SCRT-based TNT combined with PD-1 inhibitors could further improve the CR rate for LARC without unexpected serious adverse events.

Objective:To investigate the changes of upper airway and adenoids in children with hypertrophic adenoid skeletal classⅠ and class Ⅲ anterior crossbite after orthodontic treatment.Methods:From January 2021 to January 2022,155 children with skeletal class Ⅲ anterior crossbite who were treated with anterior traction in Stomatological Hospital,Shanxi Medical University were selected.They were divided into class Ⅲ normal group(72 cases)and class Ⅲ hypertrophic group(83 cases)according to whether the adenoids were hypertrophic.A total of 122 children with class Ⅰ anterior counterocclusion were treated with"2×4"treatment,which were di-vided into Class Ⅰ normal group(60 cases)and class Ⅰ hypertrophy group(62 cases).The changes of upper airway and adenoid were compared between the two groups.Results:(1)After intervention,only the width of nasopharynx cavity increased(P＜0.05)in Class Ⅰ normal group and Class Ⅰ hypertrophic group,and there was no significant difference in other indicators(P＞0.05).(2)After intervention,the adenoid thickness and A/N ratio of children with skeletal class Ⅲ anterior crossbite decreased,and the width of na-sopharynx cavity,nasopharynx airway space,soft palate upper and rear airway space,uvula tip rear airway space,the minimum air-way space between soft palate and adenoid,and mandibular plane angle increased(P＜0.05).The A/N ratio of class Ⅲ hypertrophic group was lower than that of class Ⅲ normal group(P＜0.05).Conclusion:Orthodontic treatment can reduce the degree of adenoid hypertrophy and expand the airway in children with skeletal class Ⅲ anterior crossbite.Only the width of the nasopharynx cavity can be widened after the correction intervention for children with adenoid hypertrophy and skeletal class Ⅰ anterior crossbite.

Liver cancer is a prevalent and highly malignant cancer worldwide,with 90％of cases being hepatocellular carcinoma,presenting a significant risk to human health.As early-stage liver cancer often lacks specific manifestations,most patients with liver cancer are already in the middle and late stage of the disease when liver cancer is diagnosed,thus,missing the opportunity for optimal radical treatment.However,the exploration of the treatment for middle and advanced primary hepatocellular carcinoma has never ceased.In recent years,transarterial chemoembolization(TACE)has been included in the standard treatment regimens for primary hepatocellular carcinoma.With the advancement of multidisciplinary comprehensive treatment,various treatment options to reduce the burden of liver cancer lesion with satisfactory therapeutic results have been reported and have been widely used in clinical practice,e.g.hepatic artery infusion chemotherapy(HAIC),targeted therapy,and immunotherapy in combination with TACE,which have significantly improved the overall survival of patients with liver cancer.This paper aims to make a comprehensive review about the latest progress of combination use of TACE and other therapies in reducing tumor burden and improving patient survival in the treatment of primary hepatocellular carcinoma,and to summarize the key issues in combination therapy that require more in-depth research.(J Intervent Radiol,2024,33:688-692)

ObjectiveTo study the potential quality marker （Q-marker） of Tinosporae Radix associated with efficacy of "relieving sore throat" based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS）， multivariate statistical analysis （MSA）， and network pharmacology. MethodUPLC-Q-TOF-MS was used to identify the main chemical components in 18 batches of Tinosporae Radix. On this basis， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were employed to screen out the main marker components that caused differences between groups. Moreover， network pharmacology technology was applied to predict the potential "sore throat-relieving" components， and the molecular docking between the common components resulting from MSA and network pharmacology and the core targets was carried out to verify the marker components. ResultA total of 17 compounds， including alkaloids， diterpenoid lactones， and sterols， were identified by UPLC-Q-TOF-MS. Five main differential components were found by MSA： Columbamine， jatrorrhizine， palmatine， menisperine， and columbin. Network pharmacology analysis yielded six compounds： tetrahydropalmatine， palmatine， menisperine， fibleucin， neoechinulin A， and columbin which were selected as potential "sore throat-relieving" components of Tinosporae Radix. They may relieve sore throat by acting on interleukin-6， epidermal growth factor receptor， prostaglandin G/H synthase 2， matrix metalloproteinase-9， proto-oncogene tyrosine-protein kinase Src and other targets， and regulating Hepatitis B， influenza A， human T-cell virus infection， human cytomegalovirus infection， coronavirus disease-2019， and other signaling pathways. The common active components in Tinosporae Radix resulting from MSA and network pharmacology analysis were palmatine， menisperine， and columbin， which had high binding affinity with six core targets and can be used as the Q-marker components of Tinosporae Radix in "relieving sore throat". ConclusionThis study predicts the "sore throat-relieving" Q-marker of Tinosporae Radix， which lays a basis for developing the quality standard of Tinosporae Radix based on the efficacy and improving the quality evaluation system of the medicinal.

Objective:To explore the relationship between urinary exosomal microRNA (exo-miR) - 155 in patients with type 2 diabetes mellitus (T2DM) and the onset and severity of diabetic kidney disease (DKD).Methods:From January to May 2019, 5 patients with T2DM normoalbuminuria and 5 patients with type 2 DKD were recruited from the Department of Endocrinology and Metabolism of Chongming Hospital of Shanghai Health Medical College as a microRNA screening cohort. Urine samples were collected to extract urinary exosomes, and the urine exo-miR spectrum was detected and analyzed using the miRCURY LNA array. From June 2019 to October 2022, 351 patients with T2DM who met the enrollment criteria and were matched by age and sex were included in the validation cohort in the Department of Endocrinology and Metabolism of the hospital. Patients were divided into 3 groups according to urinary albumin/creatinine ratio (UACR): normoalbuminuria group (UACR<30 mg/g, n=143), microalbuminuria group (30 mg/g≤UACR≤300 mg/g, n=171) and macroalbuminuria group (UACR>300 mg/g, n=37). According to DKD diagnostic guidelines, microalbuminuria group and macroalbuminuria group were classified into DKD group. Real-time fluorescence quantitative PCR was used to detect the expression level of exo-miR-155 in urine. Results:The results of transmission electron microscopy, nanoparticle tracking analysis, and Western blotting showed that the extraction of exosome vesicles was successful. In the screening cohort, according to the screening criteria of P<0.05 and fold changes (FC)>1.5, 226 differentially expressed miRNAs were identified from the urinary exosomes of the DKD group compared to the T2DM group. Among them, miR-155 ranged highest (FC=32.75, P<0.001). In the validation cohort, compared with the normoalbuminuria group [0.76 (0.55, 0.95)], the macroalbuminuria group [1.84 (1.18, 2.42)] had the most significant increase in urinary exo-miR-155 level ( Z=-7.411, P<0.001), followed by the microalbuminuria group [0.86 (0.69, 1.25)] ( Z=-4.092, P<0.001), and the urinary exo-miR-155 level in the macroalbuminuria group was significantly higher than that in the microalbuminuria group ( Z=-5.841, P<0.001). The correlation analysis showed that urinary exo-miR-155 level was positively correlated with UACR ( r s=0.329, P<0.001) and negatively correlated with estimated glomerular filtration rate ( r s=-0.249, P=0.015). The results of receiver operating characteristic curve analysis showed that the area under the curve of urinary exo-miR-155 level predicted DKD progression in T2DM patients was 0.892 (95% CI 0.859-0.925), corresponding cutoff value was 0.982, and the sensitivity and specificity were 71.9% and 87.7%, respectively. Multivariate logistic regression analysis showed that urinary exo-miR-155≥0.982 was an independent risk factor for progression to DKD in T2DM patients ( OR=3.310, 95% CI 1.981-5.530, P<0.001). Conclusion:The expression level of urinary exo-miR-155 is increased in T2DM patients with microalbuminuria and macroalbuminuria, which is related to the degree of albuminuria, and can be used as a predictive marker to identify potential DKD.

The online version of the original article can be found at https://doi.org/10.1631/jzus.B2000190 Erratum to: J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2020 21(10):779-795 https://doi.org/10.1631/jzus.B2000190.