BACKGROUND: Epoxyeicosatrienoic acids (EETs), which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase, are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). Many studies have revealed that sEH gene deletion exerts protective effects against diabetes. Vascular calcification is a common complication of diabetes, but the potential effects of sEH on diabetic vascular calcification are still unknown. METHODS: The level of aortic calcification in wild-type and Ephx2-/- C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining, immunohistochemistry staining, and immunofluorescence staining. Mouse vascular smooth muscle cell lines (MOVAS cells) treated with β-glycerol phosphate (0.01 mol/L) plus advanced glycation end products (50 mg/L) were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells, which was detected by Western blotting, alizarin red staining, and Von Kossa staining. RESULTS: sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice. EETs (especially 11,12-EET and 14,15-EET) efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2 (NID2) expression. Interestingly, suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products. NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification. Moreover, NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2 (IGF2) and phospho-ERK1/2 expression in MOVAS cells. Overall, sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and, then, inactivating the downstream IGF2-ERK1/2 signaling pathway. CONCLUSIONS sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels, which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.
Animals ; Mice ; Vascular Calcification/metabolism* ; Mice, Inbred C57BL ; Epoxide Hydrolases/metabolism* ; Diabetes Mellitus, Experimental/genetics* ; Male ; Gene Deletion ; MAP Kinase Signaling System/genetics* ; Cell Line ; Immunohistochemistry ; Muscle, Smooth, Vascular/metabolism* ; Signal Transduction/genetics* ; Mice, Knockout

OBJECTIVES: To explore the mechanism by which Tougu Xiaotong Capsule (TXC) promotes chondrogenic differentiation and cartilage repair in mice with osteoarthritis (OA). METHODS: Fifty 8-week-old male C57BL mice were randomly divided into normal control group, cartilage damage (induced by subchondral ring-shaped drilling) model group and TXC treatment groups at low, moderate and high doses (184, 368 and 736 mg/kg, respectively). Saline (in normal control and model groups) and TXC were administered after modeling by daily gavage for 6 consecutive weeks. The changes of cartilage damage in the mice were assessed by measuring thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) and using micro-CT, modified safranine O and fast green staining, HE staining, and qPCR. Primary cultures of mouse synovial mesenchymal stem cells (SMSCs) with lentivirus vector transfection for interfering CXCL12, TXC treatment, or both for 24 h were examined for chondrogenic differentiation using immunofluorescence staining, scratch assay, immunocytochemistry, and Western blotting. RESULTS: In mouse models with cartilage damage, TXC treatment at the moderate dose significantly alleviated joint pain, promoted cartilage repair, and upregulated the mRNA expression levels of CXCL12, GDF5, collagen II, aggrecan, Comp and Sox9 in the cartilage tissue. In primary mouse SMSCs, CXCL12 knockdown resulted in significant reduction of GDF5 protein expression, migration ability and Sox9 protein expression, and these changes were obviously reversed by TXC treatment. CONCLUSIONS TXC promotes chondrogenic differentiation of mouse SMSCs to promote repair of cartilage damage in mice by activating the CXCL12/GDF5 pathway.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Osteoarthritis/metabolism* ; Male ; Growth Differentiation Factor 5/metabolism* ; Mice, Inbred C57BL ; Mice ; Chemokine CXCL12/metabolism* ; Signal Transduction/drug effects* ; Cell Differentiation/drug effects* ; Cartilage, Articular/drug effects* ; Mesenchymal Stem Cells/cytology*

ObjectiveTo explore the potential molecular mechanism of Qizhu Kang'ai Formula （芪术抗癌方， QZKAF） for the treatment of colorectal cancer （CRC）. MethodsNetwork pharmacology was used to analyze the active ingredients and targets of QZKAF for CRC， and analyze the key targets of QZKAF for the treatment of CRC by gene function annotation （GO） and Kyoto Encyclopedia of Genomes （KEGG） pathway enrichment analysis. Molecular docking was applied to predict the binding activity of the core active ingredients to the key targets. A orthotopic transplantation tumor mice model of CRC was established to validate the key targets of QZKAF for CRC obtained from network pharmacology analysis. Forty-eight mice were randomly divided into the sham operation group， the model group， the 5-fluorouracil （5-Fu） group， and the QZKAF low-， medium-， and high-dose groups， with 8 mice in each group. Except for the sham operation group， the remaining groups underwent colon cancer orthotopic transplantation tumor modeling. The 5-Fu group was given 30 mg/kg of 5-Fu by intraperitoneal injection once every 3 days on the alternate day after modeling， while the QZKAF low-， medium-， and high-dose groups were given 2.925， 5.85， and 11.7 g/（kg·d） of QZKAF by gastric gavage， respectively， and the sham-operation group and the model group were gavaged with 0.1 ml/10 g of normal saline every day， all for 21 days. The in situ tumors mass and the number of liver metastases were compared between the groups. The pathological changes of colon tumor tissues were observed by HE staining， and the protein expression of protein tyrosine phosphatase nonreceptor type 1 （PTPN1）， vinculin， integrin subunit αν， integrin subunit β3， and E-cadherin were detected in colon tumor tissues by Western blot. ResultsNetwork pharmacology screening yielded that the top six core active ingredients of QZKAF intervening in CRC were quercetin， kaempferol， apigenin， luteolin， baicalein and ursolic acid. There were 212 targets of action， and the ranked top three were prostaglandin endoperoxide synthase 1 （PTGS1）， prostaglandin endoperoxide synthase 2 （PTGS2）， and PTPN1， which may be the key targets of QZKAF in the treatment of CRC. These key targets were significantly enriched mainly in phosphatidylinositol 3-kinase/protein kinase B （PI3K-Akt） signaling pathway， focal adhesion and adhesion junction. Molecular docking results： except for PTGS1 with better binding activity to quercetin， kaempferol， and apigenin （binding energy ≥-7.0 kcal/mol）， PTGS1 showed strong binding activity to lignans， baicalein， ursolic acid， as well as PTGS2 and PTPN1 to the six core active ingredients （binding energy ＜-7.0 kcal/mol）. Experimental validation results： the protein expression of PTPN1， vinculin， integrin subunit αν， integrin subunit β3 in the colon tumor tissues of mice in the model group significantly increased， and the expression of E-cadherin significantly decreased compared to those in the sham operation group （P＜0.05）. Compared to those in the model group， the mass of the in situ tumors was reduced， and the number of hepatic metastasis nodules decreased in the high- and medium-dose QZKAF groups （P＜0.05）； the expression levels of PTNP1， vinculin， integrin subunit αν， integrin subunit β3 and E-cadherin in all QZKAF groups and 5-Fu group showed different degrees of retracement， and the changes of the indicators in all QZKAF groups showed a certain degree of dose-dependence （P＜0.05）. HE staining showed that the nuclei of tumor cells in the model group were mostly schizophrenic， and there were different degrees of nuclear fragmentation of tumor cells in all QZKAF groups with more in the medium- and high-dose groups. ConclusionQZKAF could inhibit the growth of in situ tumors and liver metastasis of CRC. Its mechanism might be related to the regulation of tumor cell-cell and tumor cell-extracellular matrix adhesion by PTPN1.

Objective:To summarize and elucidate the impact of ambient air pollution on biological aging among middle-aged and older adults.Methods:"Air pollution""Biological age""Epigenetic age""Biological aging"and"Epigenetic aging", as well as specific names of air pollutants and biological age were used as search keywords. This study searched the databases of PubMed and Web of Science for eligible English articles and CNKI, CQVIP, Wanfang, CBM, CSTP and other Chinese databases for eligible Chinese articles from inception until June 30, 2023. The language was limited to Chinese and English.Results:Among the 14 included articles, five studies investigated the impact of air pollution on DNA methylation age using different algorithms, while six studies explored the relationship between air pollutants and telomere length. Six studies focused on frailty as an outcome, and an additional study revealed the relationship between fine particulate matter (PM 2.5) and its components with composite indicator age (KDM age). The results indicated that, although different forms of biological ages were susceptible to different ambient air pollutants at different degrees, previous studies had consistently found that the increased levels of PM 2.5 and one of its major components, black carbon (BC), could significantly accelerate the biological aging of middle-aged and older adults. Similar trends were observed with nitrogen oxides (NO x) and ozone (O 3) but with relatively limited evidence. Conclusion:Major air pollutants could accelerate the biological aging of middle-aged and older adults.

Objective:To summarize and elucidate the impact of ambient air pollution on biological aging among middle-aged and older adults.Methods:"Air pollution""Biological age""Epigenetic age""Biological aging"and"Epigenetic aging", as well as specific names of air pollutants and biological age were used as search keywords. This study searched the databases of PubMed and Web of Science for eligible English articles and CNKI, CQVIP, Wanfang, CBM, CSTP and other Chinese databases for eligible Chinese articles from inception until June 30, 2023. The language was limited to Chinese and English.Results:Among the 14 included articles, five studies investigated the impact of air pollution on DNA methylation age using different algorithms, while six studies explored the relationship between air pollutants and telomere length. Six studies focused on frailty as an outcome, and an additional study revealed the relationship between fine particulate matter (PM 2.5) and its components with composite indicator age (KDM age). The results indicated that, although different forms of biological ages were susceptible to different ambient air pollutants at different degrees, previous studies had consistently found that the increased levels of PM 2.5 and one of its major components, black carbon (BC), could significantly accelerate the biological aging of middle-aged and older adults. Similar trends were observed with nitrogen oxides (NO x) and ozone (O 3) but with relatively limited evidence. Conclusion:Major air pollutants could accelerate the biological aging of middle-aged and older adults.

Objective To investigate the mutation characteristics and influencing factors of ethambutol(EMB)resistance gene of Mycobacterium tuberculosis in Guangxi Zhuang Autonomous region,and to provide evidence for molecular diagnosis and clinical treatment of tuberculosis.Methods A total of 655 strains of Myco-bacterium tuberculosis(52 ethambutol resistant strains and 603 ethambutol sensitive strains)were collected continuously from 30 TB drug resistance monitoring sites in Guangxi in 2018-2019,and the mutation characteristics and influencing factors of ethambutol resistant genes were analyzed by whole genome sequencing.Results Among 655 strains of Mycobacterium tuberculosis,54 strains had ethambutol drug resistance gene mutation,the mutation rate was 8.24%(54/655).Among 52 EMB-resistant strains detected by proportional method,21 had gene mutation,the mutation rate was 40.38%(21/52),and 33 of 603 EMB-sensitive strains had gene mutation,the mutation rate was 5.47%(33/603).The gene mutation rate in drug-resistant strains was higher than that in sensitive strains(χ2 = 77.133,P = 0.000).The coincidence rate of EMB drug resistance phenotype and gene mutation was 40.38%(21/52),and the results of the two tests were not highly consistent(Kappa = 0.343,P<0.001).The mutant genes of 54 strains were embA,embB and embC,and there were 20 mutant forms,among which 29 were mutated at unit point,accounting for 53.70%(29/54),and 25 were mutated at joint site,accounting for 46.30%(25/54).Among the unit point mutations,embB306(35.19%)had the highest mutation proportion,followed by embB497(5.56%)and embB406(3.70%).Among the joint site mutations,embC270+embB378 had the highest mutation proportion(22.22%),The second was embB306+embA-12(3.70%).Gender,anti-tuberculosis treatment history,genotype and MDR might be related to EMB gene mutation(χ2 = 9.388,P = 0.004;χ2 = 27.084,P = 0.000;χ2 = 6.671,P = 0.010;χ2 = 68.826,P = 0.000).Multivariate logistic regression analysis showed that male(OR = 6.150),retreatment(OR = 2.636)and multidrug resistance(OR = 7.333)may be risk factors for EMB resistance gene mutation,and Beijing genotype may be a protective factor for EMB resistance gene mutation(OR = 0.511).Conclusion EMB resistance of Mycobacterium tuberculosis is related to embA,embB and embC gene mutations,and the incidence of EMB resistance phenotype is not high.For male,retreatable,MDR-resistant,and non-Beijing genotype TB patients,attention should be paid to the mutation of the EMB resistance gene.

The amygdala is an important hub for regulating emotions and is involved in the pathophysiology of many mental diseases, such as depression and anxiety. Meanwhile, the endocannabinoid system plays a crucial role in regulating emotions and mainly functions through the cannabinoid type-1 receptor (CB₁R), which is strongly expressed in the amygdala of non-human primates (NHPs). However, it remains largely unknown how the CB₁Rs in the amygdala of NHPs regulate mental diseases. Here, we investigated the role of CB₁R by knocking down the cannabinoid receptor 1 (CNR1) gene encoding CB₁R in the amygdala of adult marmosets through regional delivery of AAV-SaCas9-gRNA. We found that CB₁R knockdown in the amygdala induced anxiety-like behaviors, including disrupted night sleep, agitated psychomotor activity in new environments, and reduced social desire. Moreover, marmosets with CB₁R-knockdown had up-regulated plasma cortisol levels. These results indicate that the knockdown of CB₁Rs in the amygdala induces anxiety-like behaviors in marmosets, and this may be the mechanism underlying the regulation of anxiety by CB₁Rs in the amygdala of NHPs.
Animals ; Callithrix ; Receptors, Cannabinoid ; Anxiety ; Amygdala ; Cannabinoids ; Phenotype

Objective:To investigate the expression of B lymphocyte induced maturation protein 1 (Blimp-1) in peripheral blood CD4 + T cells, CD19 + B cells and labial glands of patients with primary Sj?gren′s syndrome (pSS) and the correlation of Blimp-1 with clinical features. Methods:Expression of PRDM1 at mRNA level in CD4 + T cells, CD19 + B cells and labial gland tissues were detected by RT-PCR. Immunohistochemistry (IHC) was used to observe the distribution of Blimp-1. Correlation of PRDM1 expression at mRNA level with clinical indicators was analyzed. Results:PRDM1 expression at mRNA level in CD4 + T and CD19 + B cells were significantly higher in pSS group than in healthy control (HC) group ( P<0.01). Based on European League Against Rheumatism Sj?gren′s Syndrome Disease Activity Index (ESSDAI), the patients were classified into two groups: active group (ESSDAI≥5) and inactive group (ESSDAI<5). PRDM1 expression at mRNA level in CD4 + T and CD19 + B cells were also higher in the active group than in inactive group ( P<0.05). Blimp-1 protein accumulated around the acinus and duct of labial gland and in the germinal center in pSS patients. PRDM1 expression at mRNA level in labial gland tissues of pSS patients was positively correlated with lymphocyte infiltration ( r=0.781, P<0.001). Conclusions:pSS displayed high expression of Blimp-1. Blimp-1 might affect pSS disease activity and have clinical significance in pSS treatment.

Clopidogrel is one of the anti-platelet drugs, which is widely used in the world.It plays an important role in the treatment of patients with acute coronary syndrome and those undergoing percutaneous coronary intervention.Clopidogrel is effective in inhibiting the activity of platelets, decreasing the incidence of thrombosis in the stent, and then reducing the risk of adverse cardiovascular events in affected individuals. However, some patients still have coronary thrombosis after taking clopidogrel.This phenomenon is known as clopidogrel resistance or clopidogrel non-response or low response. Identification of clopidogrel resistance is of great significance in preventing the occurrence of adverse cardiovascular events.This paper provides guidance for the clinical treatment of clopidogrel resistance by discussing the definition, mechanisms and laboratory evaluation of clopidogrel resistance.

Objective To investigate the effects of Sini decoction on function of hypothalamic-pituitary-adrenal axis in patients with sepsis.Methods A prospective single-blind randomized controlled trial was conducted.60 septic patients were divided into three groups with the method of random number table,20 cases in the control group,20 in the Chinese herb group,and 20 in corticoid group.All of them received routine treatment.Patients in Chinese herb group were given Sini decoction in addition (decoction of monkshood 15 g,dried ginger 15 g,honey-fried licorice 10 g) 100 mL/d orally or by nasal feeding,while patients in corticoid group were given hydrocortisone 200 mg/d intravenously instead,both for 7 days.Before the treatment,3 days and 14 days after treatment,blood was collected to determine the levels of adrenocorticotropic hormone (ACTH) and cortisol,and the result of ACTH stimulating test was observed.At the same time,acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score was recorded,and 3-day shock recovery rate and 28-day death rate were also compared among these groups.Results None of the three groups showed different result in ACTH stimulating test (x2=1.101,P=0.605).ACTH in three groups was gradually decreased.Compared with that before treatment,ACTH in Chinese herb group and corticoid groups began to decrease obviously on day 3 (ng/L:29.90 ± 3.31 vs.33.10 ±.3.31,28.20 ±.2.45 vs.33.30 ± 3.84,both P＜0.01),while in control group declined ACTH appeared later (on day 14) compared with before treatment (ng/L:29.40 ±5.63 vs.33.50 ±4.89,P＜0.05).No obvious difference in ACTH level was showed between the Chinese herb group and the cortical group (both P＞0.05).Cortisol level in both Chinese herb and cortical groups showed a raise-fall biphase trend while there was no change in the control.The cortical levels on day 3 in Chinese herb and cortical groups were much higher than that before treatment (μg/L:343.04 ± 31.20 vs.294.70 ±42.10,331.25 ±42.80 vs.280.36 ± 38.10,both P＜0.01) and that of control group (μg/L:291.61 ± 41.50,both P＜0.01),though no significant statistical difference was observed between two groups (both P＞0.05).APACHE Ⅱ score on day 14 in control,Chinese herb and cortical groups was significantly lower than that before treatment (16.8 ± 5.1 vs.20.1 ± 4.3,13.4 ± 3.2 vs.18.3 ± 3.8,15.1 ± 2.5 vs.19.5 ± 4.0,all P＜0.01),and the score was much lower in Chinese herb group comparing with that of control group (P＜0.05).No statistical difference was observed among control,Chinese herb and cortical groups in lowering 28-day death rate [35.0％ (7/20),25.0％ (5/20),20.0％ (4/20)] and improving 3-day shock recovery rate [40.0％ (8/20),70.0％ (14/20),60.0％ (12/20),all P＞0.05].Conclusions Sini decoction could elevate cortisol while lower ACTH at the early stage of sepsis.Sini decoction could also effectively improve symptoms and hypothalamic-pituitary-adrenal axis function in septic patients without affecting death rate.