Objective:To investigate whether paclitaxel (PTX) can induce immunogenic cell death (ICD) in vascular smooth muscle cells (VSMCs), and explore the new molecular mechanism of PTX-coated balloon angioplasty in intracranial atherosclerotic stenosis.Methods:(1) Cell culture and identification: VSMCs were induced into synthetic vascular smooth muscle cells (sVSMCs); the mRNA and protein expressions of smooth muscle protein 22-α (SM22-α) and α-smooth muscle actin (α-SMA) in VSMCsS and sVSMCs were detected by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Human acute monocytic leukemia cell line THP-1 was induced into dendritic cells (DCs); the CD86 and CD83 expressions in THP-1 and DCs were detected by flow cytometry. (2) Cell viability detection: cell counting kit-8 (CCK-8) assay was used to detect the cell viability of sVSMCs after 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L PTX or under 0, 50, 100, 200, 400, and 600 mmHg (1 mmHg=0.133 kPa) pressures. (3) ICD marker detection: sVSMCs were collected and divided into blank-control group, dimethyl sulfoxide (DMSO) group and PTX group (cultured with 3.2 μmol/L PTX) at normal state and pressure procedure (188 mmHg), respectively; calreticulin (CRT) expression was detected by immunofluorescent staining; adenosine triphosphate (ATP) expression was detected by luciferase assay, and high mobility group protein B1 (HMGB1) expression was detected by enzyme-linked immunosorbent assay (ELISA). (4) ICD-related immune activation assay detection: sVSMCs and DCs were collected and divided into DCs group, PTX+DCs group (cultured with 3.2 μmol/L PTX), DCs+sVSMCs group, and PTX+DCs+sVSMCs group (cultured with 3.2 μmol/L PTX); CD86 and CD83 expressions were detected by flow cytometry; interleukin (IL)-2, IL-10 and interferon-γ (IFN-γ) levels were detected by ELISA. The sVSMCs, DCs and CD8 +T cells were collected and divided into sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, sVSMCs+DCs+CD8 +T cell group, PTX+sVSMCs group (cultured with 3.2 μmol/L PTX), and PTX+sVSMCs+DCs+CD8 +T cell group (cultured with 3.2 μmol/L PTX); proliferation of these cells was detected by cell clone formation assay. Results:(1) The SM22-α and α-SMA mRNA and protein expressions in the sVSMCs group were significantly lower than those in the VSMCs group ( P<0.05); rate of double-positive CD83 and CD86 in the DCs group was significantly higher than that in the THP-1 group ( P<0.05). (2) The sVSMCs viability decreased in a concentration-dependent manner after PTX treatment at concentrations of 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L, respectively, with significant differences ( P<0.05); half maximal inhibitory concentration (IC 50) of PTX on sVSMCs was 3.2 μmol/L; no significant difference in sVSMCs viability after 3.2 μmol/L PTX treatment was noted under 0, 50, 100, 200, 400, and 600 mmHg pressures ( P>0.05). (3) Under normal state and pressure procedure, CRT fluorescent intensity of sVSMCs in the PTX group (42.00±3.50, 24.19±2.41) was significantly higher than that in the blank-control group (8.60±1.8, 8.42±1.7) and DMSO group (10.23±1.47, 9.71±1.01), ATP luminescence intensity (17 399.33±2 035.58, 17 445.67±2 449.34) was significantly higher than that in the blank-control group (9 021.33±726.84, 10 271.33±2 194.22) and DMSO group (11 977.33±960.91, 11 683.33±419.50), and HMGB1 concentration ([3 258.31±502.08] pg/mL, [3 265.27±246.06] pg/mL) was significantly higher than that in the blank-control group ([1 156.48±184.96] pg/mL, [1 205.20±196.36] pg/mL) and DMSO group ([1 309.59±75.03] pg/mL, [1 265.51±14.52] pg/mL, P<0.05). (4) The PTX+DCs+sVSMCs group had significantly higher CD83, CD86, IFN-γ and IL-2 expressions and lower IL-10 expression than the DCs group, PTX+DCs group, and DCs+sVSMCs group ( P<0.05); the PTX+sVSMCs group and PTX+sVSMCs+DCs+CD8 +T cell group had significantly lower clone formation rate compared with the sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, and sVSMCs+DCs+CD8 +T cell group ( P<0.05). Conclusion:PTX can promote ICD in VSMCs by promoting DCs activation and enhancing CD8 +T cell toxicity.

Objective:To investigate the mechanism of mitochondrial DNA (mtDNA)-reactive oxygen species (ROS)-dynamin-related protein 1 (Drp1) axis in regulating phenotypic transformation of vascular smooth muscle cells (VSMCs).Methods:(1) VSMCs were divided into a control group, a synthetic VSMCs group, and a Drp1 siRNA+synthetic VSMCs group; cells in the Drp1 siRNA+synthetic VSMCs group were transfected with 50 nmol/L Drp1 siRNA for 48 h; cells in the latter two groups were treated with 20 ng/mL platelet-derived growth factor (PDGF)-BB, while cells in the control group were treated with an equal volume of solvent. After another 24 h of culture, Drp1 expression in VSMCs, and mitochondrial Drp1 and mitofusin 2 (Mfn2) expressions were detected by Western blotting, and changes in mitochondrial morphology were detected by mitochondrial fluorescent staining. (2) VSMCs were divided into a control group, a synthetic VSMCs group, and a mitochondrial fission inhibitor 1 (Mdivi-1)+synthetic VSMCs group; cells in the Mdivi-1+synthetic VSMCs group were pretreated with 50 μmol/L Mdivi-1 for 2 h; and cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 hours of continued culture, expressions of α-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), proliferating cell nuclear antigen (PCNA), and Cyclin D1 were detected by Western blotting; invasion and migration abilities of VSMCs were detected by Transwell assay and scratch wound healing assay, respectively. (3) VSMCs were divided into a control group, a synthetic VSMCs group, and a N-acetylcysteine (NAC)+synthetic VSMCs group; cells in the NAC+synthetic VSMCs group were pretreated with 5 mmol/L NAC for 1 h; cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 h of continued culture, expressions of Drp1, phosphorylated (p)-Drp1, α-SMA, SM22-α, PCNA, and Cyclin D1 were detected by Western blotting; changes in mitochondrial morphology were detected by mitochondrial fluorescent staining; intracellular ROS level was detected by 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe; cell invasion and migration abilities were detected by Transwell assay and scratch wound healing assay, respectively. (4) VSMCs were divided into a control group, a synthetic VSMCs group, and a 5-Aza-2'-deoxycytidine (5-Aza-dC)+synthetic VSMCs group; cells in the 5-Aza-dC+synthetic VSMCs group were pretreated with 2 μmol/L 5-Aza-dC for 1 h; and then, cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 h of continued culture, agarose gel electrophoresis was used to analyze the methylation degree in the mitochondrial D-loop region; intracellular ROS level was detected using DCFH-DA fluorescent probe; expressions of mitochondrial DNMT1, α-SMA, SM22-α, PCNA, and Cyclin D1 were detected by Western blotting; invasion and migration abilities were detected by Transwell assay and scratch wound healing assay, respectively.Results:(1) Compared with the control group and synthetic VSMCs group, the Drp1 siRNA+synthetic VSMCs group had significantly decreased Drp1 protein expression ( P<0.05). Compared with the control group, the synthetic VSMCs group had significantly increased Drp1 protein expression and decreased Mfn2 protein expression in the mitochondria ( P<0.05); compared with the synthetic VSMCs group, the Drp1 siRNA+synthetic VSMCs group had statistically decreased Drp1 protein expression and increased Mfn2 protein expression in the mitochondria ( P<0.05). Results of mitochondrial fluorescent staining showed that mitochondria in the control group were with filamentous structure, while mitochondrial fission in the synthetic VSMCs group was enhanced, and morphology of mitochondria in the Drp1 siRNA+synthetic VSMCs group tended to be continuous and complete. (2) Compared with the control group, the synthetic VSMCs group had statistically decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the Mdivi-1+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster cell scratch healing; compared with the synthetic VSMCs group, the Mdivi-1+synthetic VSMCs group had smaller number of migrating cells and slower cell scratch healing. (3) Compared with the control group (1.10±0.02), the synthetic VSMCs group (1.53±0.02) had significantly increased p-Drp1 protein expression ( P<0.05). Compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group (0.90±0.02) had statistically decreased p-Drp1 protein expression ( P<0.05). Results of mitochondrial fluorescent staining showed that mitochondria in cells of the control group were in a filamentous structure, while mitochondrial fission in cells of the synthetic VSMCs group was enhanced, and morphology of mitochondria in the NAC+synthetic VSMCs group tended to be continuous and complete. Results of DCFH-DA fluorescent probe showed that ROS level in the synthetic VSMCs group was higher than that in the control group, and ROS level in the NAC+synthetic VSMCs group was lower than that in the synthetic VSMCs group. Compared with the control group, the synthetic VSMCs group had significantly decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster cell scratch healing; compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group had smaller number of migrating cells and slower cell scratch healing. (4) Results of agarose gel electrophoresis showed that compared with the control group, the synthetic VSMCs group had significantly increased methylation rate in the mitochondrial D-loop region ( P<0.05); compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had statistically decreased methylation rate in the mitochondrial D-loop region ( P<0.05). Compared with the control group, the synthetic VSMCs group had statistically increased mitochondrial DNMT1 protein expression (1.03±0.03 vs. 0.55±0.03, P<0.05); and compared with the synthetic VSMCs group, the the 5-Aza-dC+synthetic VSMCs group (0.62±0.03) had significantly decreased mitochondrial DNMT1 protein expression ( P<0.05). Results of DCFH-DA fluorescent probe showed that ROS level in the synthetic VSMCs group was higher than that in the control group; ROS level in the 5-Aza-dC+synthetic VSMCs group was lower than that in the synthetic VSMCs group. Compared with the control group, the synthetic VSMCs group had significantly decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster scratch healing. Compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had smaller number of migrating cells and slower scratch healing. Conclusion:The mtDNA-ROS-Drp1 axis may regulate the phenotypic transformation of VSMCs by modulating mitochondrial epigenetic modifications.

Objective To study the medication law of TCM compounds for ischemic stroke using data mining methods;To provide reference for clinical medication.Methods Literature about TCM treatment for ischemic stroke was retrieved from CNKI,VIP,Wanfang Data and SinoMed from May 31,2014 to May 31,2024.Drug frequency analysis,dosage analysis,property and taste attribution analysis,association rule analysis,factor analysis and clustering analysis were performed using Excel 2019,SPSS Modeler 18,SPSS 25 software.Results A total of 180 prescriptions for the treatment of ischemic stroke were included,involving 208 kinds of Chinese materia medica,with a total frequency of 1 996 times,of which 20 were high-frequency medicines(≥24 times),with Chuanxiaong Rhizoma,Angelicae Sinensis Radix,Pheretima,Astragali Radix,Paeoniae Radix Rubra and Carthami Flos being the most frequent;the efficacy of the medicines included activating blood circulation and removing blood stasis,tonifying the deficiency,pacifying the liver,calming the wind and clearing away heat;the main properties were warm,cold and neutral;the main tastes were bitter,sweet and pungent;the main meridians were liver,spleen,heart and lung meridians;among the drug dosages of drugs with frequency≥24,the mean dosage of Chuanxiong Rhizoma,Angelicae Sinensis Radix,Pheretima,Astragali Radix and Paeoniae Radix Rubra were 12.24,12.91,10.52,40.00,12.13 g,respectively.In the systematic clustering and complex network analysis,it was suggested that the core TCM prescriptions were:Xuefu Zhuyu Decoction with modified Banxia Baizhu Tianma Decoction,modified Chaihu Shugan Powder,Buyang Huanwu Decoction with modified Huangqi Guizhi Wuwu Decoction and modified Liujunzi Decoction.Conclusion TCM treatment for ischemic stroke mostly uses the methods of activating blood circulation and expelling phlegm,nourishing blood to promote blood circulation,clearing heat and cooling blood,and benefiting qi and neutralizing qi,which provides ideas and methods for following research.

Objective To study the medication law of TCM compounds for ischemic stroke using data mining methods;To provide reference for clinical medication.Methods Literature about TCM treatment for ischemic stroke was retrieved from CNKI,VIP,Wanfang Data and SinoMed from May 31,2014 to May 31,2024.Drug frequency analysis,dosage analysis,property and taste attribution analysis,association rule analysis,factor analysis and clustering analysis were performed using Excel 2019,SPSS Modeler 18,SPSS 25 software.Results A total of 180 prescriptions for the treatment of ischemic stroke were included,involving 208 kinds of Chinese materia medica,with a total frequency of 1 996 times,of which 20 were high-frequency medicines(≥24 times),with Chuanxiaong Rhizoma,Angelicae Sinensis Radix,Pheretima,Astragali Radix,Paeoniae Radix Rubra and Carthami Flos being the most frequent;the efficacy of the medicines included activating blood circulation and removing blood stasis,tonifying the deficiency,pacifying the liver,calming the wind and clearing away heat;the main properties were warm,cold and neutral;the main tastes were bitter,sweet and pungent;the main meridians were liver,spleen,heart and lung meridians;among the drug dosages of drugs with frequency≥24,the mean dosage of Chuanxiong Rhizoma,Angelicae Sinensis Radix,Pheretima,Astragali Radix and Paeoniae Radix Rubra were 12.24,12.91,10.52,40.00,12.13 g,respectively.In the systematic clustering and complex network analysis,it was suggested that the core TCM prescriptions were:Xuefu Zhuyu Decoction with modified Banxia Baizhu Tianma Decoction,modified Chaihu Shugan Powder,Buyang Huanwu Decoction with modified Huangqi Guizhi Wuwu Decoction and modified Liujunzi Decoction.Conclusion TCM treatment for ischemic stroke mostly uses the methods of activating blood circulation and expelling phlegm,nourishing blood to promote blood circulation,clearing heat and cooling blood,and benefiting qi and neutralizing qi,which provides ideas and methods for following research.

Objective:To investigate whether paclitaxel (PTX) can induce immunogenic cell death (ICD) in vascular smooth muscle cells (VSMCs), and explore the new molecular mechanism of PTX-coated balloon angioplasty in intracranial atherosclerotic stenosis.Methods:(1) Cell culture and identification: VSMCs were induced into synthetic vascular smooth muscle cells (sVSMCs); the mRNA and protein expressions of smooth muscle protein 22-α (SM22-α) and α-smooth muscle actin (α-SMA) in VSMCsS and sVSMCs were detected by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Human acute monocytic leukemia cell line THP-1 was induced into dendritic cells (DCs); the CD86 and CD83 expressions in THP-1 and DCs were detected by flow cytometry. (2) Cell viability detection: cell counting kit-8 (CCK-8) assay was used to detect the cell viability of sVSMCs after 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L PTX or under 0, 50, 100, 200, 400, and 600 mmHg (1 mmHg=0.133 kPa) pressures. (3) ICD marker detection: sVSMCs were collected and divided into blank-control group, dimethyl sulfoxide (DMSO) group and PTX group (cultured with 3.2 μmol/L PTX) at normal state and pressure procedure (188 mmHg), respectively; calreticulin (CRT) expression was detected by immunofluorescent staining; adenosine triphosphate (ATP) expression was detected by luciferase assay, and high mobility group protein B1 (HMGB1) expression was detected by enzyme-linked immunosorbent assay (ELISA). (4) ICD-related immune activation assay detection: sVSMCs and DCs were collected and divided into DCs group, PTX+DCs group (cultured with 3.2 μmol/L PTX), DCs+sVSMCs group, and PTX+DCs+sVSMCs group (cultured with 3.2 μmol/L PTX); CD86 and CD83 expressions were detected by flow cytometry; interleukin (IL)-2, IL-10 and interferon-γ (IFN-γ) levels were detected by ELISA. The sVSMCs, DCs and CD8 +T cells were collected and divided into sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, sVSMCs+DCs+CD8 +T cell group, PTX+sVSMCs group (cultured with 3.2 μmol/L PTX), and PTX+sVSMCs+DCs+CD8 +T cell group (cultured with 3.2 μmol/L PTX); proliferation of these cells was detected by cell clone formation assay. Results:(1) The SM22-α and α-SMA mRNA and protein expressions in the sVSMCs group were significantly lower than those in the VSMCs group ( P<0.05); rate of double-positive CD83 and CD86 in the DCs group was significantly higher than that in the THP-1 group ( P<0.05). (2) The sVSMCs viability decreased in a concentration-dependent manner after PTX treatment at concentrations of 0, 0.01, 0.05, 0.5, 5, 10, 50, and 100 μmol/L, respectively, with significant differences ( P<0.05); half maximal inhibitory concentration (IC 50) of PTX on sVSMCs was 3.2 μmol/L; no significant difference in sVSMCs viability after 3.2 μmol/L PTX treatment was noted under 0, 50, 100, 200, 400, and 600 mmHg pressures ( P>0.05). (3) Under normal state and pressure procedure, CRT fluorescent intensity of sVSMCs in the PTX group (42.00±3.50, 24.19±2.41) was significantly higher than that in the blank-control group (8.60±1.8, 8.42±1.7) and DMSO group (10.23±1.47, 9.71±1.01), ATP luminescence intensity (17 399.33±2 035.58, 17 445.67±2 449.34) was significantly higher than that in the blank-control group (9 021.33±726.84, 10 271.33±2 194.22) and DMSO group (11 977.33±960.91, 11 683.33±419.50), and HMGB1 concentration ([3 258.31±502.08] pg/mL, [3 265.27±246.06] pg/mL) was significantly higher than that in the blank-control group ([1 156.48±184.96] pg/mL, [1 205.20±196.36] pg/mL) and DMSO group ([1 309.59±75.03] pg/mL, [1 265.51±14.52] pg/mL, P<0.05). (4) The PTX+DCs+sVSMCs group had significantly higher CD83, CD86, IFN-γ and IL-2 expressions and lower IL-10 expression than the DCs group, PTX+DCs group, and DCs+sVSMCs group ( P<0.05); the PTX+sVSMCs group and PTX+sVSMCs+DCs+CD8 +T cell group had significantly lower clone formation rate compared with the sVSMCs group, sVSMCs+DCs group, sVSMCs+CD8 +T cell group, and sVSMCs+DCs+CD8 +T cell group ( P<0.05). Conclusion:PTX can promote ICD in VSMCs by promoting DCs activation and enhancing CD8 +T cell toxicity.

Objective:To investigate the mechanism of mitochondrial DNA (mtDNA)-reactive oxygen species (ROS)-dynamin-related protein 1 (Drp1) axis in regulating phenotypic transformation of vascular smooth muscle cells (VSMCs).Methods:(1) VSMCs were divided into a control group, a synthetic VSMCs group, and a Drp1 siRNA+synthetic VSMCs group; cells in the Drp1 siRNA+synthetic VSMCs group were transfected with 50 nmol/L Drp1 siRNA for 48 h; cells in the latter two groups were treated with 20 ng/mL platelet-derived growth factor (PDGF)-BB, while cells in the control group were treated with an equal volume of solvent. After another 24 h of culture, Drp1 expression in VSMCs, and mitochondrial Drp1 and mitofusin 2 (Mfn2) expressions were detected by Western blotting, and changes in mitochondrial morphology were detected by mitochondrial fluorescent staining. (2) VSMCs were divided into a control group, a synthetic VSMCs group, and a mitochondrial fission inhibitor 1 (Mdivi-1)+synthetic VSMCs group; cells in the Mdivi-1+synthetic VSMCs group were pretreated with 50 μmol/L Mdivi-1 for 2 h; and cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 hours of continued culture, expressions of α-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), proliferating cell nuclear antigen (PCNA), and Cyclin D1 were detected by Western blotting; invasion and migration abilities of VSMCs were detected by Transwell assay and scratch wound healing assay, respectively. (3) VSMCs were divided into a control group, a synthetic VSMCs group, and a N-acetylcysteine (NAC)+synthetic VSMCs group; cells in the NAC+synthetic VSMCs group were pretreated with 5 mmol/L NAC for 1 h; cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 h of continued culture, expressions of Drp1, phosphorylated (p)-Drp1, α-SMA, SM22-α, PCNA, and Cyclin D1 were detected by Western blotting; changes in mitochondrial morphology were detected by mitochondrial fluorescent staining; intracellular ROS level was detected by 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe; cell invasion and migration abilities were detected by Transwell assay and scratch wound healing assay, respectively. (4) VSMCs were divided into a control group, a synthetic VSMCs group, and a 5-Aza-2'-deoxycytidine (5-Aza-dC)+synthetic VSMCs group; cells in the 5-Aza-dC+synthetic VSMCs group were pretreated with 2 μmol/L 5-Aza-dC for 1 h; and then, cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 h of continued culture, agarose gel electrophoresis was used to analyze the methylation degree in the mitochondrial D-loop region; intracellular ROS level was detected using DCFH-DA fluorescent probe; expressions of mitochondrial DNMT1, α-SMA, SM22-α, PCNA, and Cyclin D1 were detected by Western blotting; invasion and migration abilities were detected by Transwell assay and scratch wound healing assay, respectively.Results:(1) Compared with the control group and synthetic VSMCs group, the Drp1 siRNA+synthetic VSMCs group had significantly decreased Drp1 protein expression ( P<0.05). Compared with the control group, the synthetic VSMCs group had significantly increased Drp1 protein expression and decreased Mfn2 protein expression in the mitochondria ( P<0.05); compared with the synthetic VSMCs group, the Drp1 siRNA+synthetic VSMCs group had statistically decreased Drp1 protein expression and increased Mfn2 protein expression in the mitochondria ( P<0.05). Results of mitochondrial fluorescent staining showed that mitochondria in the control group were with filamentous structure, while mitochondrial fission in the synthetic VSMCs group was enhanced, and morphology of mitochondria in the Drp1 siRNA+synthetic VSMCs group tended to be continuous and complete. (2) Compared with the control group, the synthetic VSMCs group had statistically decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the Mdivi-1+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster cell scratch healing; compared with the synthetic VSMCs group, the Mdivi-1+synthetic VSMCs group had smaller number of migrating cells and slower cell scratch healing. (3) Compared with the control group (1.10±0.02), the synthetic VSMCs group (1.53±0.02) had significantly increased p-Drp1 protein expression ( P<0.05). Compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group (0.90±0.02) had statistically decreased p-Drp1 protein expression ( P<0.05). Results of mitochondrial fluorescent staining showed that mitochondria in cells of the control group were in a filamentous structure, while mitochondrial fission in cells of the synthetic VSMCs group was enhanced, and morphology of mitochondria in the NAC+synthetic VSMCs group tended to be continuous and complete. Results of DCFH-DA fluorescent probe showed that ROS level in the synthetic VSMCs group was higher than that in the control group, and ROS level in the NAC+synthetic VSMCs group was lower than that in the synthetic VSMCs group. Compared with the control group, the synthetic VSMCs group had significantly decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster cell scratch healing; compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group had smaller number of migrating cells and slower cell scratch healing. (4) Results of agarose gel electrophoresis showed that compared with the control group, the synthetic VSMCs group had significantly increased methylation rate in the mitochondrial D-loop region ( P<0.05); compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had statistically decreased methylation rate in the mitochondrial D-loop region ( P<0.05). Compared with the control group, the synthetic VSMCs group had statistically increased mitochondrial DNMT1 protein expression (1.03±0.03 vs. 0.55±0.03, P<0.05); and compared with the synthetic VSMCs group, the the 5-Aza-dC+synthetic VSMCs group (0.62±0.03) had significantly decreased mitochondrial DNMT1 protein expression ( P<0.05). Results of DCFH-DA fluorescent probe showed that ROS level in the synthetic VSMCs group was higher than that in the control group; ROS level in the 5-Aza-dC+synthetic VSMCs group was lower than that in the synthetic VSMCs group. Compared with the control group, the synthetic VSMCs group had significantly decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster scratch healing. Compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had smaller number of migrating cells and slower scratch healing. Conclusion:The mtDNA-ROS-Drp1 axis may regulate the phenotypic transformation of VSMCs by modulating mitochondrial epigenetic modifications.

Here, we report a previously unrecognized syndromic neurodevelopmental disorder associated with biallelic loss-of-function variants in the RBM42 gene. The patient is a 2-year-old female with severe central nervous system (CNS) abnormalities, hypotonia, hearing loss, congenital heart defects, and dysmorphic facial features. Familial whole-exome sequencing (WES) reveals that the patient has two compound heterozygous variants, c.304C>T (p.R102*) and c.1312G>A (p.A438T), in the RBM42 gene which encodes an integral component of splicing complex in the RNA-binding motif protein family. The p.A438T variant is in the RRM domain which impairs RBM42 protein stability in vivo. Additionally, p.A438T disrupts the interaction of RBM42 with hnRNP K, which is the causative gene for Au-Kline syndrome with overlapping disease characteristics seen in the index patient. The human R102* or A438T mutant protein failed to fully rescue the growth defects of RBM42 ortholog knockout ΔFgRbp1 in Fusarium while it was rescued by the wild-type (WT) human RBM42. A mouse model carrying Rbm42 compound heterozygous variants, c.280C>T (p.Q94*) and c.1306_1308delinsACA (p.A436T), demonstrated gross fetal developmental defects and most of the double mutant animals died by E13.5. RNA-seq data confirmed that Rbm42 was involved in neurological and myocardial functions with an essential role in alternative splicing (AS). Overall, we present clinical, genetic, and functional data to demonstrate that defects in RBM42 constitute the underlying etiology of a new neurodevelopmental disease which links the dysregulation of global AS to abnormal embryonic development.
Female ; Animals ; Mice ; Humans ; Child, Preschool ; Intellectual Disability/genetics* ; Heart Defects, Congenital/genetics* ; Facies ; Cleft Palate ; Muscle Hypotonia

A large number of non-specific immune cells, such as macrophages, neutrophils and natural killer cells, as well as specific immune cells, such as B cells and T cells, are presented in the atherosclerotic plaques and arterial blood. In addition, immune-related regulatory molecules such as interferon-γ, interleukin-17, and interleukin-1β highly express in atherosclerotic lesion tissues, which is similar to the tumor immune microenvironment and influence the atherosclerosis progression. In this paper, the research progress of immune cells and related factors involved in the immune microenvironment around cerebral atherosclerotic lesions is reviewed to provide idea and basis for its clinical diagnosis and treatment.

Objective:To explore the mechanism of Fuyuan Xingnao Decoction in treatment of cerebral infarction based on network pharmacology and molecular docking.Methods:The active components and action targets of Fuyuan Xingnao Decoction were screened by using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP),Traditional Chinese Medicine Integrated Database (TCMID),Bioactivity data of small organic molecules (PubChem),Universal Protein (Uniprot) and Swiss Target Prediction database platform. The databases of GeneCards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and Drug Bank and Pharmacogenomics Knowledgebase (PharmGKB) were used to screen targets of cerebral infarction. The drug target genes in Fuyuan Xingnao Decoction were intersected with those of cerebral infarction, the intersecting targets were introduced into Cytoscape 3.8.2 software to construct the component target network, and the PPI protein interaction network was constructed by using STRING analysis platform and Cytoscape 3.8.2 software to screen the core targets. Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) function enrichment analysis were carried out on the common target genes of Fuyuan Xingnao Decoction and cerebral infarction disease to obtain the relevant signal pathways. Finally, AutoDock and Pymol software were used for molecular docking between the predicted target and its corresponding components.Results:After screening, 80 effective components of Fuyuan Xingnao Decoction for treatment of cerebral infarction and 214 common targets of Fuyuan Xingnao Decoction and cerebral infarction were obtained. The core targets such as MAPK1, RELA, TP53, JUN, AKT1 and HSP90AA1 were related to the key targets of cerebral infarction, and they participated in the biological process of regulating the response to drugs, lipopolysaccharide and oxygen level, etc. The cell composition involved membrane raft, membrane micro region and nerve cell body, etc. Molecular functions mainly focused on nuclear receptor activity, ligand activated transcription factor activity, DNA binding transcription factor binding, etc.; it also involved in signal pathway of lipid and atherosclerosis, chemical carcinogen and receptor activation, fluid shear stress and atherosclerosis, etc. Molecular docking showed that good binding activities were seen between Quercetin and HSP90AA1 (-9.4 kJ/mol), between Kaempferol and HSP90AA1 (-9.4 kJ/mol), between Isorhamnetin and HSP90AA1 (-9.1 kJ/mol), and between Quercetin and JUN (-8.6 kJ/mol).Conclusion:Fuyuan Xingnao Decoction can prevent and treat cerebral infarction by regulating vascular endothelial function, promoting blood circulation, repairing and improving neural function, protecting blood-brain barrier, reducing cell apoptosis, and regulating immune and inflammatory response.

Objective To observe the image characteristics ofmultispectral scanning laser imaging (MSLI) and OCT in patients with pregnancy induced hypertension syndrome (PIHS).Methods A total of 112 patients (224 eyes) of PIHS patients diagnosed in Obstetrics Department of Tianjin First Central Hospital from May 2016 to May 2017 were included in this study.The average age of the patients was 27.00±2.14 years.The average course of the disease was 15.00 ±8.27 days.There were 174 eyes in 87 patients of blurred vision,dazzling and visual fatigue consciously.All patients performed BCVA,direct ophthalmoscope,B ultrasound,confocal scanning laser Ophthalmoscope (cSLO) and spectral-domain OCT (SD-OCT).SD-OCT was performed with Spectralis HRA+OCT from Heidelberg Company in Germany to acquire tomographic images.Using Herdelberg's colorful program (MultiColor) based on cSLO and operating in accordance with standard methods,one scan simultaneously obtained blue light reflection based on 488 nm,green light reflection based on 515 nm,and infrared reflection based on 820 nm,synthesis to MSLI.Fundus abnormalities were classified into arterial spasm (stage Ⅰ),arteriosclerosis (stage Ⅱ),and retinopathy (stage Ⅲ).OCT examination was divided into normal and abnormal cases according to the abnormality of retinal morphology and thickness.Results Of the 224 eyes,68 eyes (30.36％) showed normal fundus examination and 156 eyes (69.64％) showed abnormal fundus performance.Among them,28 eyes were stage Ⅰ (17.95％);40 eyes were stage Ⅱ (25.64％);88 eyes were stage Ⅲ (56.41％).Thirty-six eyes (16.07％) showed normal fundus and 188 eyes (83.93％) showed abnormal performance with OCT.Of the 188 eyes with abnormal fundus performance,86 eyes (45.74％) had retinal neuroepithelial serous detachment;56 eyes (29.79％) had RPE detachment;optic disc edema,bulge,and local reflexes in the retinal nerve fiber layer were enhanced and/or the thickness increased in 46 eyes (24.47％).In MSLI,48 eyes (21.43％) showed normal fundus;176 eyes (78.57％) showed abnormal performance.Retinal edema was showed in green on MSLI,serous retinal neuroepithelial layer detachment,RPE layer detachment,retinal nerve fiber layer thickening,accompanied by changes in local retinal structure.The higher the degree of bulge,the darker the color.Consistent with the range of retinal edema revealed by SD-OCT.Conclusions MSLI and SD-OCT images show highly consistent lesions in PIHS patients.MSLI can more clearly show superficial and deep retinal lesions.