OBJECTIVES: To investigate the structural changes of rat thoracic aorta and changes in expression levels of Bmal1 and cyclins in thoracic aorta endothelial cells following heat stress. METHODS: Twenty male SD rats were randomized equally into control group and heat stress group. After exposure to 32 ℃ for 2 weeks in the latter group, the rats were examined for histopathological changes and Bmal1 expression in the thoracic aorta using HE staining and immunohistochemistry. In the cell experiments, cultured rat thoracic aortic endothelial cells (RTAECs) were incubated at 40 ℃ for 12 h with or without prior transfection with a Bmal1-specific small interfering RNA (si-Bmal1) or a negative sequence. In both rat thoracic aorta and RTAECs, the expressions of Bmal1, the cell cycle proteins CDK1, CDK4, CDK6, and cyclin B1, and apoptosis-related proteins Bax and Bcl-2 were detected using Western blotting. TUNEL staining was used to detect cell apoptosis in rat thoracic aorta, and the changes in cell cycle distribution and apoptosis in RTAECs were analyzed with flow cytometry. RESULTS: Compared with the control rats, the rats exposed to heat stress showed significantly increased blood pressures and lowered heart rate with elastic fiber disruption and increased expressions of Bmal1, cyclin B1 and CDK1 in the thoracic aorta (P<0.05). In cultured RTAECs, heat stress caused significant increase of Bmal1, cyclin B1 and CDK1 protein expression levels, which were obviously lowered in cells with prior si-Bmal1 transfection. Bmal1 knockdown also inhibited heat stress-induced increase of apoptosis in RTAECs as evidenced by decreased expression of Bax and increased expression of Bcl-2. CONCLUSIONS Heat stress upregulates Bmal1 expression and causes alterations in expressions of cyclins to trigger apoptosis of rat thoracic aorta endothelial cells, which can be partly alleviated by suppressing Bmal1 expression.
Animals ; ARNTL Transcription Factors/genetics* ; Male ; Aorta, Thoracic/metabolism* ; Rats ; Rats, Sprague-Dawley ; Endothelial Cells/metabolism* ; Apoptosis ; Cells, Cultured ; Heat-Shock Response ; Cyclin B1/metabolism* ; CDC2 Protein Kinase/metabolism* ; Cyclins/metabolism* ; RNA, Small Interfering ; bcl-2-Associated X Protein/metabolism*

Corticotomy is a clinical procedure to accelerate orthodontic tooth movement characterized by the regional acceleratory phenomenon (RAP). Despite its therapeutic effects, the surgical risk and unclear mechanism hamper the clinical application. Numerous evidences support macrophages as the key immune cells during bone remodeling. Our study discovered that the monocyte-derived macrophages primarily exhibited a pro-inflammatory phenotype that dominated bone remodeling in corticotomy by CX3CR1^CreERT2; R26^GFP lineage tracing system. Fluorescence staining, flow cytometry analysis, and western blot determined the significantly enhanced expression of binding immunoglobulin protein (BiP) and emphasized the activation of sensor activating transcription factor 6 (ATF6) in macrophages. Then, we verified that macrophage specific ATF6 deletion (ATF6^f/f; CX3CR1^CreERT2 mice) decreased the proportion of pro-inflammatory macrophages and therefore blocked the acceleration effect of corticotomy. In contrast, macrophage ATF6 overexpression exaggerated the acceleration of orthodontic tooth movement. In vitro experiments also proved that higher proportion of pro-inflammatory macrophages was positively correlated with higher expression of ATF6. At the mechanism level, RNA-seq and CUT&Tag analysis demonstrated that ATF6 modulated the macrophage-orchestrated inflammation through interacting with Tnfα promotor and augmenting its transcription. Additionally, molecular docking simulation and dual-luciferase reporter system indicated the possible binding sites outside of the traditional endoplasmic reticulum-stress response element (ERSE). Taken together, ATF6 may aggravate orthodontic bone remodeling by promoting Tnfα transcription in macrophages, suggesting that ATF6 may represent a promising therapeutic target for non-invasive accelerated orthodontics.
Animals ; Mice ; Macrophages/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Tooth Movement Techniques/methods* ; Activating Transcription Factor 6/metabolism* ; Bone Remodeling ; Flow Cytometry ; Blotting, Western

ObjectiveTo explore the underlying mechanism by which the Chinese medicine compound Yitangkang granule（YTK） treats diabetic kidney disease （DKD） by observing its effects on podocyte autophagy through the regulation of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/forkhead transcription factor O1 （FoxO1） signaling pathway mediated by silent information regulator 1 （SIRT1） via advanced glycation end products （AGE）/receptor for AGE （RAGE） axis. MethodNinety-six 8-week-old healthy male SPF-grade Wistar rats were selected and randomly divided into blank control group （B）， model control group， high-dose YTK （40 g·kg^-1）， medium-dose YTK （20 g·kg^-1）， low-dose YTK （10 g·kg^-1）， and Western medicine control （20 mg·kg^-1 losartan） groups. The DKD rat model was established by high-fat diet feeding combined with intraperitoneal injection of streptozotocin. After successful modeling， the rats in each group received the corresponding treatments for eight weeks. The levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， and catalase （CAT） were measured according to the instructions of the respective assay kits. Hematoxylin and eosin （HE） staining was used to observe pathological changes in kidney tissues. Immunohistochemistry was employed to detect the average optical density values of α-smooth muscle actin （α-SMA）， fibronectin （FN）， desmin， and nephrin. Western blot analysis was used to measure the expression levels of PI3K， phosphorylated PI3K （p-PI3K）， Akt， phosphorylated Akt （p-Akt）， RAGE， SIRT1， Caspase-3， and FoxO1 proteins in kidney tissues of DKD rats. ResultCompared with the blank control group， the model group showed significantly lower levels of SOD， GSH-Px， and CAT， and significantly higher levels of MDA （P<0.01）. The rats exhibited severe kidney damage. The positive expression of podocyte marker proteins α-SMA， FN， and desmin increased significantly， while nephrin and podocin significantly decreased （P<0.01）. The expression levels of PI3K， p-PI3K， Akt， p-Akt， RAGE， and Caspase-3 proteins were significantly elevated， while SIRT1 and FoxO1 protein levels were significantly reduced （P<0.01）. Compared with the model control group， rats in the YTK treatment groups showed significantly higher levels of SOD， GSH-Px， and CAT， and significantly lower levels of MDA in serum （P<0.01）. The degree of kidney damage was reduced to varying extents. The average optical density values of podocyte marker proteins α-SMA， FN， and desmin were significantly decreased， while nephrin and podocin significantly increased （P<0.01）. The expression levels of PI3K， p-PI3K， Akt， p-Akt， RAGE， and Caspase-3 in kidney tissues were significantly reduced， while SIRT1 and FoxO1 expression levels significantly increased （P<0.01）. The Chinese medicine groups demonstrated a clear dose-response trend. ConclusionYTK may alleviate kidney pathological damage， reduce proteinuria， and protect kidney function in DKD rats， thereby delaying the progression of DKD by improving podocyte autophagy through the AGE-RAGE axis-mediated SIRT1 regulation of the PI3K/Akt/FoxO1 signaling pathway. Additionally， a dose-response relationship was observed in the Chinese medicine groups.

ObjectiveTo observe the effects of Yuejuwan in the treatment of psychological and heart diseases （PHD） and explore its mechanism. MethodThirty 6-week-old healthy male SPF AopE^-/- mice and 10 homologous C57BL/6J mice were selected for the experiment. The 30 AopE^-/- mice were divided into a model group， low-dose （7.58 g·kg^-1·d^-1） and high-dose （30.32 g·kg^-1·d^-1） Yuejuwan groups， with 10 mice in each group， and 10 C57BL/6J mice were assigned to the blank control group. Intragastrical administration lasted 12 weeks. During feeding， the PHD model was induced by chronic unpredictable mild stress （CUMS） combined with high-fat diet in mice. After intragastric administration， the behavioral results ［open field test （OFT） and sucrose preference test （SPT）］ of mice in each group， the content of aspartic transaminase （AST）， alanine aminotransferase （ALT）， 5-hydroxytryptamine （5-HT）， noradrenaline （NE）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， total cholesterol （TC）， and triglyceride （TG） in serum of mice detected by the automatic biochemical analyzer， the oil red O staining and HE staining of aorta and liver and Masson staining of myocardial tissues were used for model evaluation. Finally， liver TMT-labeled quantitative proteomics was used to explore the mechanism of action. ResultThe model mice showed obvious manifestations of depression， anxiety， loss of interest， and despair， manifest lipid deposition in the aorta and liver by pathological observation， and increased myocardial fibrosis in myocardial tissues. After intragastric administration of Yuejuwan， the above symptoms and indexes of the PHD model mice were improved. Compared with the blank control group， the model group showed decreased standing times， cumulative time in the central area， total moving distance， moving speed， and sucrose preference at week 12 （P<0.01）. Compared with the model group， the Yuejuwan groups showed decreased indexes mentioned above （P<0.01）. After sample collection， AST， ALT， and TG levels in the model group were higher （P<0.01） and the levels of 5-HT， NE， and HDL-C were lower than those in the blank control group （P<0.01）. The results of liver TMT labeled quantitative proteomics suggested that the PHD model mainly caused the changes in protein expression levels such as ApoE， UGT1A5， and FASN in mice，involving acetyl CoA metabolism，response to bacteria，cellular amino acid catabolism， and other processes，which were related to the abnormal metabolic function of the liver. The efficacy of Yuejuwan against PHD was achieved mainly through the regulation of high mobility group nucleosomal-binding domain 2 （HMGN2）， CALD1， and Mup7 protein expression levels and correcting the biological processes and abnormal pathways related to the pathogenesis of PHD，including muscle contraction，tight junction pathway，myocardial contraction pathway，and focal adhesion pathway. ConclusionCUMS combined with high-fat diet is reasonable in the induction of the PHD model in AopE^-/- mice. Yuejuwan can correct the depression and anxiety conditions of PHD model mice，reduce the aortic plaque， and recover the abnormal blood lipid and liver function levels. Furthermore， Yuejuwan can correct abnormal biological processes and pathways of PHD model mice. The differential proteins screened throughout the experiment and the involved physiological and pathological changes are the focus of the next experiment.

ObjectiveTo determine the mechanism of Yitangkang in correcting excessive apoptosis of skeletal muscle cells to improve insulin resistance （IR） by inhibiting the advanced glycation end product （AGE）/receptor for the advanced glycation end product （RAGE） signaling pathway. Method① In vitro experiments. Yitangkang-medicated serum was prepared. C2C12 cells were divided into a blank group， a model group， high-， medium-， and low-dose Yitangkang-medicated serum groups （40， 20， and 10 g·kg^-1）， and a RAGE inhibitor group. The IR model was induced by palmitic acid in C2C12 cells except for those in the blank group. After the corresponding intervention methods were conducted，the cell viability and glucose consumption level of each group were determined. In addition，the apoptosis rate was determined using flow cytometry. The mRNA and protein expression levels of the important apoptotic proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， p53， cysteinyl aspartate-specific protease-3 （Caspase-3）， and cysteinyl aspartate-specific protease-9 （Caspase-9）］ were determined using Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. ② In vivo experiments. Ninety-six eligible Wistar rats were divided into a blank group， a model group， high-，medium-，and low-dose Yitangkang groups （40， 20， and 10 g·kg^-1）， and a western medicine group （pioglitazone hydrochloride，1.35 mg·kg^-1）. The IR model was induced using high-glucose and high-fat feed for diabetes combined with intraperitoneal injection of low-dose streptozotocin （STZ） in animals and verified by the hyperinsulinemic-euglycemic clamp （HEC） test. After the model was determined successfully， the rats in each group were given intragastric administration of drugs as required. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was performed to determine the number of positive apoptotic cells in the skeletal muscle tissues of rats in each group，while Real-time polymerase chain reaction（Real-time PCR） and Western blot were performed to determine the mRNA and protein expression levels of the important apoptotic proteins Bcl-2， Bax， p53， Caspase-3， and Caspase-9. Result① In vitro experiments. compared with the blank group， the model groups showed increased apoptosis rate of C2C12 cells and decreased cell viability and glucose consumption （P<0.01）. Compared with the model group， the Yitangkang-medicated serum groups and the RAGE inhibitor group showed decreased apoptosis rate of C2C12 cells and increased cell viability and glucose consumption （P<0.01）. Compared with the blank group， the model group showed decreased expression levels of Bcl-2 mRNA and protein in C2C12 cells and increased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.01）. Compared with the model group， the Yitangkang-medicated serum groups and the RAGE inhibitor group showed increased expression levels of Bcl-2 mRNA and protein in C2C12 cells （P<0.01） and decreased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.05， P<0.01）. ② In vivo experiments. The number of positive apoptotic cells in the skeletal muscle tissues of rats in the model group significantly increased as compared with that in the blank group （P<0.01）. The number of positive apoptotic cells in the skeletal muscle tissues of rats in the Yitangkang groups and the western medicine group decreased as compared with that in the model group （P<0.01）. Compared with the blank group， the model group showed decreased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats and increased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.01）. Compared with the model group， the Yitangkang groups and the western medicine group showed increased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats （P<0.01） and decreased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.05， P<0.01）. The medium-dose Yitangkang showed a similar effect as RAGE inhibitor， and the effect was equivalent to that of pioglitazone hydrochloride. ConclusionYitangkang can inhibit skeletal muscle cell apoptosis by inhibiting the AGE/RAGE signaling pathway.

ObjectiveTo compare the therapeutic effects of oral Chinese medicines （including Chinese patent medicines） on coronary artery disease （CAD） by the Bayesian network Meta-analysis. MethodThe randomized controlled trials of treating CAD with oral Chinese medicines were retrieved from the China National Knowledge Infrastructure （CNKI）， Wanfang Data， VIP， PubMed， Web of Science， Embase， and Cochrane Library from the inception to December 1， 2022. The Cochrane risk of bias assessment tool was used to evaluate the quality of the included articles. The direct meta-analysis was performed to compare the performance of oral Chinese medicines alone and in combination with Western medicine in the treatment of CAD in terms of intima-media thickness （IMT）， vascular endothelial function， plaque score， hypersensitive C-reactive protein （hs-CRP）， total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， and total response rate. Furthermore， the Bayesian network Meta-analysis was performed to compare the therapeutic effects of different Chinese medicines. ResultA total of 41 articles were included. The direct meta-analysis results showed that Chinese medicines combined with Western medicine outperformed Western medicine alone in recovering all the indicators of CAD. The Bayesian network meta-analysis yielded the following results. In terms of the total response rate， modified Huangqi Guizhi Wuwutang and Sanqi Huayu pills had obvious advantages over other Chinese medicines. In terms of IMT and plaque score， Xiaoban Huazhuo decoction， Yiqi Tongluo formula， Ruangan Jiangzhi capsules， and Guanxin Shutong capsules had obvious advantages over other Chinese medicines. In terms of blood lipid indicators， Shenqi Roumai mixture， Ruangan Jiangzhi capsules， Xiaoban Huazhuo decoction， Qiwei Sanxiong decoction， and Sanqi Huayu pills were superior to other Chinese medicines. The Chinese medicines above mainly had the functions of activating blood， resolving stasis， resolving phlegm， and dredging vessels. ConclusionThe combination of oral Chinese medicines and Western medicine is effective in treating CAD. Clinicians can use the drugs targeting abnormal indicators according to the results of this Bayesian network meta-analysis combined with the actual situation of patients to achieve better therapeutic effects.

Ferroptosis is a novel iron-dependent mode of programmed cell death characterized by iron deposition and accumulation of lipid peroxidation. More and more studies have found that ferroptosis is closely related to the pathogenesis of type 2 diabetes mellitus （T2DM）. The yin-fire theory is an important part of LI Gao's spleen-stomach theory， and it is believed that qi-fire imblance and yin-fire internal generation is the main pathogenesis of T2DM. Abnormal iron metabolism may be an important prerequisite for T2DM yin-fire internal generation， while oxidative stress is the specific manifestation of T2DM qi-fire imbalance. Reactive oxygen species （ROS） is the end product of qi-fire imbalance， and lipid peroxide is the pathological products of T2DM yin-fire internal generation. This study intends to explore the pathological mechanism of qi-fire imbalance and yin-fire internal generation from the perspectives of iron metabolism， oxidative stress and lipid peroxidation， enriching the modern connotation of yin-fire theory， and benefiting traditional Chinese medicine to target against ferroptosis， and prevent and treat T2DM precisely.

OBJECTIVE：To obs erve the protective effect of protoca techuic aldehyde（PAL）on neurovascular unit （NVU） homeostasis damage in rats after cerebral ischemia-reperfusion injury （CIRI）. METHODS ：SD rats were randomly divided into sham operation group ，model group ，PAL high-dose and low-dose groups （10，20 mg/kg），with 11 rats in each group. Administration groups were given relevant medicine intragastrically. Sham operation group and model group were given the same volume of normal saline intragastrically ，10 mL/kg once a day ，for 5 days. After last administration ，CIRI model was induced by suture method ；the ultrastructural changes of NVU were observed by transmission electron microscope. Western blot assay was used to detect the expression of NUV related proteins （MAP-2，GFAP，AQP-4）in cerebral tissue. Immunofluorescence staining was used to observe the positive expression of above proteins in cerebral cortex. RESULTS ：Compared with sham operation group ， blood-brain barrier （BBB）structure of model group was destroyed severely ，the vascular lumen became narrower ，lateral edema of endothelial cells was severe ，and the thickness of basement membrane varied ；the nuclei of neurons were pyknosis and there was a large area of edema in the surrounding tissues ；the structure of glial cells was seriously damaged ，the cell body was shrunk and organelles were lost ；protein expression （or positive expression ）of MAP- 2 in brain tissue （or cerebral cortex ）were significantly decreased （P＜0.05 or P＜0.01），while protein expression （or positive expression ） of GFAP and AQP- 4 were increased significantly（P＜0.01）. After PAL intervention ，the rats had less BBB damage ，and the morphology of vascular lumen and basement membrane were not completely destroyed ；the damage of neurons was alleviated ，the pyknosis of neurons was decreased ， the chromatin was homogeneous and the heterochromatin was decreased；the damage of glial cell structure was alleviated protein expression of GFAP and AQP- 4（except for low-dose group） in cerebral tissue and positive expression of MAP- 2 and GFAP protein in cerebral cortex were reversed @qq.com significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：PAL can protect the stability of NVU from damage in CIRI model rats； the mechanism may be related to up-regulating the expression of MAP- 2 protein in cerebral cortex and down-regulating the expression of GFAP and AQP- 4 protein in brain tissue.

Objective:To evaluate the recurrence and progression of patients with pT1 high grade urothelial carcinoma of bladder (UCB) and glandular differentiation.Methods:We retrospectively analyzed the clinical and pathological information of 208 patients diagnosed as pT1 high grade urothelial carcinoma in the Fifth Central Hospital of Tianjin from January 2006 to February 2019.Among them, 78 cases were diagnosed as glandular differentiation (UCGD), the other 130 patients without histologic variants were served as control. The UCGD group included 62 male and 16 female, whose median age was 67 years old (range 38-81 years old). The control group contained 105 male and 25 female, whose median age was 66 years old (range 40-82 years old). Kaplan-Meier and Cox proportional hazard regression analyses were used to evaluate the predictors of oncologic outcomes.Results:The disease recurrence rate and progression rate in UCGD group were 65.4% (51/78) and 28.2% (22/78), higher than 38.5%(50/130) and 14.6%(19/130) of control group ( P<0.05). The median recurrence time in UCGD group was 41 months while 55 months in the control group. The median progression time in UCGD group was 39 months while 54 months in the control group. According to the univariate analysis, largest tumor size ( P=0.030), UCGD ( P=0.003) and lymphovascular invasion (LVI) ( P=0.032) were associated with disease recurrence. UCGD ( P=0.036) and LVI ( P=0.011) were associated with progression. Additionally, Cox multivariate analysis revealed that UCGD ( P=0.001), LVI ( P=0.038) were the independent factors of disease recurrence. UCGD ( P=0.007) and LVI ( P=0.037) were also found to be the independent factors of disease progression. Conclusions:Patients with T1 stage UCB and UCGD are at higher risk of disease recurrence and progression. Therefore, these patients should be followed up closely after being diagnosed and undergo individual treatment according to the situation.

Objective:To evaluate the recurrence and progression of patients with pT1 high grade urothelial carcinoma of bladder (UCB) and glandular differentiation.Methods:We retrospectively analyzed the clinical and pathological information of 208 patients diagnosed as pT1 high grade urothelial carcinoma in the Fifth Central Hospital of Tianjin from January 2006 to February 2019.Among them, 78 cases were diagnosed as glandular differentiation (UCGD), the other 130 patients without histologic variants were served as control. The UCGD group included 62 male and 16 female, whose median age was 67 years old (range 38-81 years old). The control group contained 105 male and 25 female, whose median age was 66 years old (range 40-82 years old). Kaplan-Meier and Cox proportional hazard regression analyses were used to evaluate the predictors of oncologic outcomes.Results:The disease recurrence rate and progression rate in UCGD group were 65.4% (51/78) and 28.2% (22/78), higher than 38.5%(50/130) and 14.6%(19/130) of control group ( P<0.05). The median recurrence time in UCGD group was 41 months while 55 months in the control group. The median progression time in UCGD group was 39 months while 54 months in the control group. According to the univariate analysis, largest tumor size ( P=0.030), UCGD ( P=0.003) and lymphovascular invasion (LVI) ( P=0.032) were associated with disease recurrence. UCGD ( P=0.036) and LVI ( P=0.011) were associated with progression. Additionally, Cox multivariate analysis revealed that UCGD ( P=0.001), LVI ( P=0.038) were the independent factors of disease recurrence. UCGD ( P=0.007) and LVI ( P=0.037) were also found to be the independent factors of disease progression. Conclusions:Patients with T1 stage UCB and UCGD are at higher risk of disease recurrence and progression. Therefore, these patients should be followed up closely after being diagnosed and undergo individual treatment according to the situation.