ObjectiveThis paper aims to conduct the feature analysis and correlation analysis on the ocular collateral features and differential metabolites in patients with chronic hepatitis B （CHB） complicated by glucose metabolism disorder （GMD），particularly those with the damp-heat syndrome type，by integrating shadowless scleral imaging and metabolomics technologies. MethodsA total of 313 patients were recruited from the Hepatology and Endocrinology Outpatient Departments of Public Health Clinical Center of Chengdu according to the inclusion/exclusion criteria，and they were divided into a CHB group and a CHB complicated by GMD groups （damp-heat syndrome group and non-damp-heat syndrome group）. All patients underwent high-definition ocular image acquisition and feature extraction using an intelligent analysis system for shadowless scleral imaging to analyze the differences in the counting of morphological feature scores of ocular collaterals among groups. By using a digital sampling method，24 patients from each group were randomly selected，along with 20 healthy volunteers，for untargeted metabolomic analysis of peripheral serum. Differential metabolites were identified，statistically analyzed，and subjected to potential biomarker analysis and pathway enrichment. Spearman method was performed to conduct the correlation analysis on the differential ocular collateral features and differential metabolites，followed by correlation network construction. ResultsCompared with those in the CHB group，patients with CHB complicated by GMD showed significant changes in ocular collateral feature scores such as "hillock"，"blood vessels"，and "pale dusky coloration" （P<0.05）. In comparison with those in the healthy group，metabolites including N-acetylglucosamine，acetylhomoserine，and myo-inositol （AUC>0.7） were identified as potential biomarkers for the disease. Compared with those in the CHB complicated by GMD group with non-damp-heat syndrome，patients with damp-heat syndrome exhibited significant changes in feature scores of "plaques"，"yellow coloration"，"spleen"，and "gallbladder" （P<0.05）. In comparison with those in the healthy group，metabolites such as O²′-4a-cyclic tetrahydrobiopterin，theobromine，xanthurenic acid，and L-glutamic acid 5-phosphate （AUC>0.7） were identified as potential biomarkers for the damp-heat syndrome type. The Spearman correlation analysis reveals weak to moderate linear correlations between the differential scleral collateral features and metabolites. By constructing a "disease-syndrome" network of ocular diagnosis and metabolites，"xanthurenic acid-gallbladder" and "theobromine-plaque/yellow coloration" were identified as specific molecular-phenotypic correlated biomarker clusters for CHB complicated by GMD with dampness-heat syndrome. ConclusionPatients with CHB complicated by GMD demonstrate differential ocular diagnostic features and serum metabolites corresponding to disease states and dampness-heat syndrome. These objective biomarkers can guide both clinical syndrome differentiation and medication. The macro-micro integration based on ocular feature clusters and potential metabolic biomarkers offers an innovative approach to a combined traditional Chinese and Western medicine diagnosis and treatment model for this disease.

ObjectiveTo explore the improving effect of Huangqi Chifengtang（HCT） on atherosclerosis（AS）， and elucidate its mechanism in relation to adenosine monophosphate-activated protein kinase（AMPK）/peroxisome proliferator-activated receptor α（PPARα） signaling pathway and nucleotide-binding oligomerization domain（NOD）-like receptor thermal protein domain associated protein 3（NLRP3） inflammasome. MethodsEight C57BL/6J mice were set as the normal group， and 32 ApoE^-/- mice were randomly divided into the model group， the positive drug group（atorvastatin， 5 mg·kg^-1·d^-1）， HCT low- and high-dose groups（1.95， 3.90 g·kg^-1·d^-1）. ApoE^-/- mice were fed with high-fat and high-cholesterol feed to establish an AS mouse model. After modeling， they were orally administered corresponding dose of drugs for 28 days， while the normal and model groups received an equal volume of physiological saline via oral gavage. Hematoxylin-eosin（HE） staining was used to observe the pathological status of the aorta and liver in mice， Biochemical testing and enzyme-linked immunosorbent assay（ELISA） were used to detect the levels of total cholesterol（TC）， triglycerides（TG）， low-density lipoprotein cholesterol（LDL-C）， alanine aminotransferase（ALT）， aspartate aminotransferase（AST）， C-reactive protein（CRP）， interleukin（IL）-1β， IL-18 in the serum， as well as superoxide dismutase（SOD）， malondialdehyde（MDA）， and reduced glutathione（GSH） in the liver. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to measure the mRNA expression levels of NLRP3， apoptosis-associated speck-like protein（ASC）， cysteinyl aspartate specific proteinase-1（Caspase-1）， Toll-like receptor 4（TLR4） in the aorta， and fatty acid synthase（FAS）， stearoyl-CoA desaturase 1（SCD1）， PPARα， and carnitine palmitoyltransferase 1A（CPT1A） in the liver. Immunohistochemistry was used to determine the protein expressions of NLRP3， Caspase-1， and ASC in the aorta， and Western blot was used to measure the protein expressions of AMPK， p-AMPK， sterol regulatory element binding protein-1c（SREBP-1c）， CPT1A， and FAS in the liver. ResultsCompared with the normal group， the model group showed a significant increase in lipid plaque deposition in the aorta and lipid accumulation in the liver， the levels of TC， TG， LDL-C， AST， ALT， IL-1β， IL-18 and CRP in the serum were significantly increased（P<0.01）， and the mRNA and protein expressions of aortic TLR4， NLRP3， Caspase-1 and ASC were significantly upregulated（P<0.01）. The levels of SOD and GSH in the liver were significantly reduced， while the level of MDA was significantly increased（P<0.01）. The mRNA expressions of FAS and SCD1 in the liver were significantly downregulated， while the mRNA expressions of PPARα and CPT1A were significantly upregulated. The protein expressions of p-AMPK/AMPK and CPT1A in the liver were significantly reduced， while the expressions of SREBP-1c and FAS proteins were significantly increased（P<0.01）. Compared with the model group， the low- and high-dose HCT groups showed significant improvements in aortic plaques and hepatic lipid deposition. The levels of TC， LDL-C， AST， IL-1β and IL-18 in the serum of the low-dose HCT group， as well as TC， TG， LDL-C， AST， ALT， IL-1β， IL-18 and CRP in the serum of the high-dose HCT group， were significantly reduced（P<0.01）. The mRNA expressions of TLR4， NLRP3 and Caspase-1 in the aorta of the low-dose HCT group， as well as TLR4， NLRP3， Caspase-1 and ASC in the aorta of the high-dose HCT group， were significantly downregulated（P<0.01）. The protein expressions of Caspase-1 and ASC in the aorta of the low-dose HCT group， as well as NLRP3， Caspase-1 and ASC in the high-dose HCT group， were significantly downregulated（P<0.01）. The levels of SOD and GSH in the liver of the low- and high-dose HCT groups were significantly increased， while the level of MDA in the high-dose HCT group was significantly decreased（P<0.05， P<0.01）. In the HCT-treated group， the mRNA expressions of FAS and SCD1 in the liver were significantly upregulated， while the mRNA expressions of PPARα and CPT1A were significantly downregulated， the protein expressions of p-AMPK/AMPK and CPT1A in the liver were significantly increased， while the protein expressions of SREBP-1c and FAS were significantly decreased（P<0.05， P<0.01）. ConclusionHCT can improve lipid metabolism by activating the AMPK/PPARα pathway and inhibit NLRP3 inflammasome-mediated inflammatory responses， thereby reducing hepatic lipid deposition and AS plaque formation.

Traditional Chinese medicine contains a variety of volatile components （VOCs） such as terpenes and aliphatics, which have significant pharmacological activities, and are commonly analyzed by gas chromatography （GC） and gas chromatography-mass spectrometry （GC-MS）. Although the separation ability is strong, the samples need to be pre-treated and the analysis time is long. In recent years, the combination of GC and ion migration spectrometry （IMS） has been widely used in the academic community. The combination of high separation ability of GC and rapid reaction ability of IMS has the advantages of no sample pretreatment, fast analysis time, low detection limit and simple operation. It is increasingly used in traditional Chinese medicine research. The working principle of GC-IMS was introduced, the application in VOCs research of traditional Chinese medicine was summarized, and the future development direction was prospected in this paper.

Objective:This study aimed to compare postoperative pain between minimally invasive coronary artery bypass grafting (MICS-CABG) and conventional CABG for multivessel coronary artery disease, comparing baseline characteristics and perioperative data between the two groups, and analyzing risk factors influencing postoperative pain.Methods:A total of 545 patients undergoing elective off-pump coronary artery bypass grafting (OPCABG) for multivessel coronary artery disease at Beijing Anzhen Hospital from July 2022 to July 2023 were included. There were 397 patients in the conventional CABG group (289 males, 108 females, aged 35-77 years) and 148 patients in the minimally invasive CABG group (121 males, 27 females, aged 37-84 years), with 148 patients in each group after propensity score matching. Pain levels were assessed using the Numeric Rating Scale (NRS) at the first 5 days postoperatively (acute postoperative pain, APP) and at 3, 6, and 12 months postoperatively (chronic post-surgical pain, CPSP). Logistic regression was used to analyze risk factors for CPSP at 3 months postoperatively in both groups. Results:Within 48 hours postoperatively, both groups reported maximum NRS pain intensities at rest (NRS 4.0 vs. 6.0) and during activity (NRS 5.2 vs. 7.5). From the third day after surgery, there were no significant differences in resting pain intensity between the two groups, and from the fourth day after surgery, there were no significant differences in pain intensity during movement. With 60.2% in the conventional group and 92.6% in the minimally invasive group experiencing moderate to severe pain at rest (NRS ≥ 4), and 83.5% in the conventional group and 98.0% in the minimally invasive group experiencing moderate to severe pain during activity (NRS ≥ 4). Immediately after drain removal, there was a significant reduction in pain intensity at rest in the minimally invasive group (pre-drain removal NRS 6.0 vs. post-drain removal NRS 2.7), compared to the conventional group (pre-drain removal NRS 4.0 vs. post-drain removal NRS 2.3). However, there was no significant difference in the reduction of pain intensity during activity between the minimally invasive group (pre-drain removal NRS 7.5 vs. post-drain removal NRS 4.2) and the conventional group (pre-drain removal NRS 6.0 vs. post-drain removal NRS 2.7). At 3 months postoperatively, the incidence of CPSP was 35.9% in the conventional group and 35.1% in the minimally invasive group. At 6 months postoperatively, the incidence of CPSP was significantly lower compared to 3 months in both groups (conventional group 8.7% vs. minimally invasive group 6.8%, P<0.001). In the conventional group, higher Europe SCORE Ⅱ was identified as an independent risk factor for CPSP at 3 months postoperatively, while in the minimally invasive group, higher BMI and postoperative use of flurbiprofen for rescue analgesia were identified as independent risk factors. Conclusion:In patients undergoing minimally invasive coronary artery bypass grafting (CABG), the early postoperative acute pain intensity and incidence were higher than those in the conventional CABG group. After drain removal, there were no significant differences in resting pain intensity between the two groups, but pain intensity during movement remained higher in the minimally invasive group compared to the conventional group. The incidence of chronic pain did not differ between the two groups but decreased significantly from 3 months postoperatively. Conventional CABG patients with high preoperative Europe SCORE Ⅱ scores, high preoperative BMI and severe postoperative acute pain tend to have more chronic pain after minimally invasive bypass surgery.

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: Whether elevation in plasma levels of amyloid and tau protein biomarkers are better indicators of cognitive decline than higher baseline levels in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD) remains understudied. Methods: We included 67 participants with twice testing for AD-related plasma biomarkers via immunomagnetic reduction (IMR) assays (amyloid beta [Aβ]1-40, Aβ1-42, total tau [t-Tau], phosphorylated tau [p-Tau] 181, and alpha-synuclein [α-Syn]) and the Mini-Mental State Examination (MMSE) over a 1-year interval. We examined the correlation between biomarker levels (baseline vs. longitudinal change) and annual changes in the MMSE scores. Receiver operating characteristic curve analysis was conducted to compare the biomarkers. Results: After adjustment, faster cognitive decline was correlated with lower baseline levels of t-Tau (β=0.332, p=0.030) and p-Tau 181 (β=0.369, p=0.015) and rapid elevation of t-Tau (β=-0.330, p=0.030) and p-Tau 181 levels (β=-0.431, p=0.004). However, the levels (baseline and longitudinal changes) of Aβ1-40, Aβ1-42, and α-Syn were not correlated with cognitive decline. aMCI converters had lower baseline levels of p-Tau 181 (p=0.002) but larger annual changes (p=0.001) than aMCI non-converters. The change in p-Tau 181 levels showed better discriminatory capacity than the change in t-Tau levels in terms of identifying AD conversion in patients with aMCI, with an area under curve of 86.7% versus 72.2%. Conclusion We found changes in p-Tau 181 levels may be a suitable biomarker for identifying AD conversion.

The structure of intestinal tissue is complex. In vitro simulation of intestinal structure and function is important for studying intestinal development and diseases. Recently, organoids have been successfully constructed and they have come to play an important role in biomedical research. Organoids are miniaturized three-dimensional (3D) organs, derived from stem cells, which mimic the structure, cell types, and physiological functions of an organ, making them robust models for biomedical research. Intestinal organoids are 3D micro-organs derived from intestinal stem cells or pluripotent stem cells that can successfully simulate the complex structure and function of the intestine, thereby providing a valuable platform for intestinal development and disease research. In this article, we review the latest progress in the construction and application of intestinal organoids.
Organoids/cytology* ; Intestines/physiology* ; Humans ; Animals ; Pluripotent Stem Cells

Objective:This study aims to investigate the expression of uridine diphosphate-glucose[]pyrophos-phorylase 2(UGP2)in breast cancer(BC)tissues and its oncogenic mechanism,assessing its potential value as a diag-nostic and prognostic biomarker for breast cancer.Methods:(1)Online database analysis was conducted to assess UGP2 mRNA and protein expression levels in breast cancer and explore their correlation with clinical characteristics.Im-munohistochemistry(IHC)was used to verify UGP2 expression in human breast cancer tumor tissues and evaluate its relationship with clinicopathological features.(2)Kaplan-Meier survival analysis and COX regression models were used to analyze the impact of UGP2 expression on breast cancer patient prognosis.(3)Bioinformatics methods were em-ployed to investigate the correlation between UGP2 and tumor immune cell infiltration,and to predict the biological func-tions and associated signaling pathways of UGP2 in breast cancer.Results:(1)The mRNA and protein expression levels of UGP2 were upregulated in breast cancer tissues(both P<0.05),and were negatively correlated with ER-positive and PR-positive status(OR<1,P<0.05),while positively correlated with Ki-67 levels and the triple-negative breast cancer(TNBC)subtype(OR>1,P<0.05).(2)Elevated expression levels of UGP2 were associated with poorer survival rates in breast cancer patients(both P<0.05)and were identified as an independent adverse prognostic factor for breast cancer(HR=1.40,P<0.05).(3)Functional analysis results suggested that UGP2 may promote tumor progression by regulating metabolism,hormone signaling,and the immune microenvironment.Additionally,UGP2 expression was negatively cor-related with NK cell activation status and positively correlated with the inhibitory state.Conclusion:UGP2 expression is elevated in breast cancer tissues and is closely associated with poor patient prognosis.It may promote cancer pro-gression through mechanisms such as metabolic reprogramming and immune suppression.UGP2 shows promise as a potential biomarker and therapeutic target in breast cancer,providing a basis for personalized treatment.

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.