Osteoprotegerin (OPG) is a soluble decoy receptor for receptor activator of nuclear factor kB ligand (RANKL), and is implicated in the pathogenesis of atherosclerosis. The aim of the present study was to examine the hypothesis that serum OPG concentrations are increased in patients with stable coronary artery disease (CAD) at different serum levels of soluble RANKL (sRANKL). The study used a case-control design in which consecutively hospitalized individuals were recruited. Fasting blood samples were taken upon admission for serum testing. Participants with previously diagnosed CAD that was asymptomatic or had controlled symptoms constituted the stable CAD group, whereas patients with negative coronary computed tomography angiography results constituted the control non-CAD group. Exclusion criteria included recent acute coronary syndrome, severe heart failure, CAD-complicating autoimmune, blood or thyroid diseases, cancer, elevated temperature with or without infection, severe liver or kidney dysfunction, abnormal calcium metabolism, recent surgery and trauma history. A total of 118 individuals were included in the study. Smoothed plots generated using the recursive method and multivariate models showed that the incidence of stable CAD increased with serum OPG level up to the turning point of 18 pg/ml. This trend was observed at both high [odds ratio (OR), 1.61; 95% confidence interval (CI), 1.04-2.50; P=0.032) and low sRANKL concentrations (OR, 1.52; 95% CI, 1.06-2.17; P=0.022) after adjustment for cardiovascular risk factors. In conclusion, serum OPG levels <= 18 pg/ml are positively associated with stable CAD, regardless of sRANKL levels. In addition, at the same serum OPG level, higher sRANKL levels are associated with a greater incidence of stable CAD compared with lower sRANKL levels. This study identified the relationship between OPG, sRANKL, and stable CAD, and established the reference range for future clinical use.

Objective:To investigate the role and significance of nuclear factor-kappa B (NF-κB) ligand receptor in the transformation of osteoid cells through the Wingless-related integration site (Wnt)/β-catenin pathway.Methods:Aortic smooth muscle cells (SMC) were isolated from SD rats and induced into osteoblast like cells. Osteoblast like cells were treated with LiCl, LiCl+ bone protection element (OPG, 0.1 ng/ml), LiCl+ OPG (1.0 ng/ml), LiCl+ OPG (10.0 ng/ml) and LiCl+ OPG (100.0 ng/ml) respectively. The control group was not treated with LiCl or OPG, and the concentration of LiCl was 20 mmol/L. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of OPG and RANKL. The von Kossa staining, calcium content, alkaline phosphatase (ALP) activity, osteocalcin and Western blotting were used to detect the degree of calcification. Immunohistochemistry and Western blotting were used to detect the activation of Wnt/β-catenin pathway. One-way ANOVA was used to compare the differences between the experimental groups and control group, LSD- t test was used for multiple comparison. Results:The expression of OPG in LiCl group (4.35±0.88) was slightly higher than that in osteoblast like cell group (4.12±0.82, t=0.603, P>0.05). The expression of RANKL in LiCl group (2.61±0.42) was significantly higher than that in osteoblast like cell group (1.52±0.35, t=2.317, P<0.05). The ratio of OPG/RANKL in LiCl group (1.67±0.41) was significantly lower than that in osteoblast like cell group (2.71±0.48, t=2.542, P<0.05). In the transformation experiment of osteoblast like cells, the calcium content, ALP activity and osteocalcin level in osteoblast like cells group and the LiCl+ OPG groups [(25.2±5.8, 68.5±13.3, 61.1±12.8, 57.6±11.3, 56.3±11.4), (80.4±10.2, 141.2±17.6, 125.7±15.3, 123.5±15.9, 122.7±15.1), (0.61±0.10, 0.82±0.14, 0.79±0.13, 0.72±0.12, 0.73±0.13)] were significantly reduced as compared with those in the LiCl group [(99.5±16.5), (186.0±39.3), (0.98±0.15), ( t=5.971, 2.795, 2.810, 2.829, 2.831, P<0.05), ( t=5.623, 2.597, 2.765, 2.791, 2.793, P<0.05), ( t=4.931, 2.379, 2.384, 2.391, 2.392, P<0.05)]. There was no significant difference in the calcium content, ALP activity and osteocalcin level among the LiCl+ OPG groups ( P>0.05), but the calcium content, ALP activity and osteocalcin level in LiCl+ OPG groups were significantly higher than those in osteoblast like cells group [( t=5.362, 2.769, 2.635, 2.633, P<0.05), ( t=5.105, 2.758, 2.732, 2.733, P<0.05), ( t=2.762, 2.569, 2.566, 2.563, P<0.05)]. The expression level of β-catenin in LiCl group (1.75±0.26) was significantly higher than that in osteoblast like cell group and LiCl+ OPG groups (1.32±0.18, 1.25±0.17, 1.25±0.16, 1.22±0.16), ( t=4.442, 2.650, 2.654, 2.712, 2.709, P<0.05), and that in LiCl+ OPG groups was significantly higher than that in osteoblast like cell group ( t=2.492, 2.455, 2.439, 2.437, P<0.05). There was no significant difference among the LiCl+ OPG groups ( P>0.05). Conclusion:Activation of Wnt/β-catenin pathway promotes the transformation of osteoid cells by RANKL-dependent and non-RANKL-dependent mechanisms.

Backgrounnd: The impact of Alzheimer's disease (AD) on the living quality and life expectancy has becoming increasingly severe, but the pathogenesis of AD is still being studied .This study aimed to investigate whether Family with sequence similarity 3 member C (FAM3C), which has been reported to be possibly associated with cognitive function, was associated with the incidence of AD.Methods: A total of 282 participants from Alzheimer’s Disease Neuroimaging Initiative (ADNI) were included. The demographic data, cerebrospinal fluid (CSF) FAM3C content, neuropsychological scores, CSF amyloid protein level, CSF tau protein level, and imaged data were collected. One-way analysis of variance and chi-square test were used to compare demographic data, neuropsychological test scores, CSF protein levels, and imaging data of three groups. Partial correlation analysis and multiple linear regression analysis were used to investigate the relationship between FAM3C and AD diagnostic indicators in different dimensions. An ordered multi-classification logistic regression model was established to determine whether FAM3C is related to the onset of AD. Results: Lower levels of FAM3C were observed in AD and mild cognitive impairment (MCI) groups. CSF FAM3C level was related to whole-brain atrophy and temporal lobe atrophy, worse cognitive performance, decreased amyloid β 1-42 (Aβ1-42), and lower regional cerebral glucose metabolism. Conclusion: CSF FAM3C might be a potential diagnostic biomarker of AD. Further study on concrete mechanisms may contribute to early diagnosis and treatment of AD.

In this study, the hypothesis that Wnt/β-catenin pathway is involved in the arterial calcification by regulating the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) system was tested. The β-catenin expression was measured in the warfarin-induced calcified arteries and the osteoblast-like cells differentiating from smooth muscle cells (SMCs) by immunohistochemistry and Western blotting. The Wnt/β-catenin pathway was activated or inhibited by lithium chloride (LiCl) or dickkopf 1 (DKK1) in vitro and in vivo. Then the calcification level was determined by von Kossa staining, Ca2+ content assay, and alkaline phosphatase (ALP) activity assay. The expression levels of osteocalcin, OPG and RANKL were detected by Western blotting or real-time PCR. The results showed that in calcified arteries and OBL cells, the activation of Wnt/β-catenin pathway significantly enhanced the calcification as evidenced by increased von Kossa stains, Ca2+ contents, ALP activities, and osteocalcin expression levels (P<0.05), and it promoted the RANKL expression (P<0.05), but slightly affected the OPG expression. These results indicated that the activation of Wnt/β-catenin pathway worsens the arterial calcification, probably by promoting the RANKL expression.

The initiation and progression of atherosclerotic cardiovascular disease (ASCVD) has always been associated with a series of risk factors. Evidences of statin therapy from randomized clinical trials are abundant, whereas discussions regarding patients with ASCVD without evidence-based risk factors are rare. Here, we describe a case of a 58-year-old woman who was diagnosed with ASCVD with none of these evidence-based risk factors. After four years of medical interventions, including atorvastatin, the patient recovered completely from severe chest pain with significant regression of atherosclerotic plaques in coronary arteries.

Heart failure (HF) is the end stage of various kinds of cardiovascular diseases and leads to a high mortality worldwide. Numerous studies have demonstrated that frequencies of CD4+CD25+Foxp3+ regulatory T cells (Tregs) are reduced in HF patients and properly expanding Tregs attenuates HF progression. Histone deacetylase (HDAC) 9 has been revealed to contribute to several cardiovascular and cerebrovascular diseases. Plenty of studies showed that HDAC9 negatively regulated the number and function of Tregs. Thus, we aim to investigate the expression of HDAC 9 in patients with chronic heart failure (CHF) and the relationship among HDAC9, Tregs and CHF. Our research showed a reduced number of Tregs and an increased expression of HDAC9 mRNA in CHF patients. Patients with CHF were divided into two groups by heart function grade of New York Heart Association (NYHA), we found that the HDAC9 mRNA expression level in NYHA grade II–III group were lower than that in NYHA grade IV group. More importantly, the correlation study suggested that the expression of HDAC9 mRNA was negatively correlated to Tregs frequency and left ventricular ejection fraction (LVEF), whereas positively correlated to larger left ventricular end-diastolic dimension (LVEDD) and B-type natriuretic peptide (BNP) in patients with CHF. The correlation studies also showed a positive correlation between HDAC9 and the severity of CHF. Our research suggests that HDAC9 may be a new indicator for assessing CHF and it may offer a new direction for research of CHF.

Objective To investigate the mechanism that receptor activator of NF-κB ligand (RANKL) promotes arterial calcification.Methods Firstly,RANKL was added into the culture media,in which the monocyte precursor cells alone were cultured.Morphological observation and tartrate resistant acid phosphatase(TRAP)stain were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells.During arterial calcification,both in vivo and in vitro expressions of RANKL and osteoprotegerin (OPG,as RANKL inhibitor)were measured via real-time PCR.The extent of osteoclast-like cell differentiation was also assessed.Results It was found that RANKL could induce osteoclast-like cell differentiation.There were no both in vivo and in vitro expressions of osteoclast-like cells in the early stage of calcification.At that time,the ratio of RANKL to OPG was very low.In the late stage of calcification,a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG.According to the results,the ratio of RANKL to OPG was very low during most of the arterial calcification period.This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation.The ratio of RANKL to OPG was (0.36 ± 0.08) (F =36) and (1.68 ± 0.08) (F =36) respectively in the early and late subgroup of calcification group in the animal model,but was zero in the control group(both P＜0.05).The ratio of RANKL to OPG was(0.42±0.09) (F=16)and(1.50 ± 0.10)(F=16)respectively in the early and late subgroup of calcification group in the cell model,but was zero in the control group(both P＜0.05).Conclusions Our result likely explains why RANKL has the ability to induce osteoclast-like cell differentiation,but acts as a promoter of calcification.

Coronary artery disease (CAD) is a multifactorial disease in which inflammation plays a central role. This study aimed to investigate the association of inflammatory markers such as the neutrophil to lymphocyte ratio (NLR), the Global Registry of Acute Coronary Events (GRACE) score with in-hospital mortality of elderly patients with acute myocardial infarction (AMI) in an attempt to explore the prognostic value of these indices for elderly AMI patients. One thousand consecutive CAD patients were divided into two groups based on age 60. The laboratory and clinical characteristics were assessed retrospectively by reviewing the medical records. The NLR and GRACE score were calculated. In the elderly (≥60 years), patients with non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI) had significantly higher NLR than did those with unstable angina (UA) and stable angina pectoris (SAP) (P<0.01). The NLR was considerably elevated in older AMI patients compared with their younger counterparts (<60 years) (P<0.05). In elderly AMI patients, the NLR was considerably higher in the high-risk group than in both the low-risk and medium-risk groups based on the GRACE score (P<0.05 and P<0.01, respectively), and the NLR was positively correlated with the GRACE score (r=0.322, P<0.001). Either the NLR level or the GRACE score was significantly higher in the death group than in the surviving group (P<0.05). By curve receiver operator characteristic curve (ROC) analysis, the optimal cut-off levels of 9.41 for NLR and 174 for GRACE score predicted in-hospital death [ROC area under the curve (AUC) 0.771 and 0.787, respectively, P<0.001]. It was concluded that an elevated NLR is a potential predictor of in-hospital mortality in elderly patients with AMI.

Background/Aims: Baicalin has been shown to be effective for various animal models of cardiovascular diseases, such as pulmonary hypertension, atherosclerosis and myocardial ischaemic injury. However, whether baicalin plays a role in cardiac hypertrophy remains unknown. Here we investigated the protective effects of baicalin on cardiac hypertrophy induced by pressure overload and explored the potential mechanisms involved. Methods: C57BL/6J-mice were treated with baicalin or vehicle following transverse aortic constriction or Sham surgery for up to 8 weeks, and at different time points, cardiac function and heart size measurement and histological and biochemical examination were performed. Results: Mice under pressure overload exhibited cardiac dysfunction, high mortality, myocardial hypertrophy, increased apoptosis and fibrosis markers, and suppressed cardiac expression of PPARα and PPARβ/δ. However, oral administration of baicalin improved cardiac dysfunction, decreased mortality, and attenuated histological and biochemical changes described above. These protective effects of baicalin were associated with reduced heart and cardiomyocyte size, lower fetal genes expression, attenuated cardiac fibrosis, lower expression of profibrotic markers, and decreased apoptosis signals in heart tissue. Moreover, we found that baicalin induced PPARα and PPARβ/δ expression in vivo and in vitro. Subsequent experiments demonstrated that long-term baicalin treatment presented no obvious cardiac lipotoxicity. Conclusions: The present results demonstrated that baicalin attenuates pressure overload induced cardiac dysfunction and ventricular remodeling, which would be due to suppressed cardiac hypertrophy, fibrosis, apoptosis and metabolic abnormality.

Objective To study the function of statins on the arterial calcification and its effect on the osteoprotegerin/receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL).Methods To constitute the calcified animal and cell models respectively,and each model was divided randomly into the calcified group,statins group,and control group.In the cell model,for the calcified group,β-glycerophosphate and Vitamin C was added into the culture medium,the statins group was given both β-glycerophosphate,Vitamin C and atorvastatin;In the animal model,the calcified group was taken the intragastric administration with Warfarin,the statins group was taken the intragastric administration with both Warfarin and atorvastatin.Ca2+ content assay,alkaline phosphatase (ALP) activity assay and osteocalcin assayed with WestemBlot were used to check the level of animal and cell calcification;real-time quantitative polymerase chain reaction (Real-time PCR) was used to check the expression of osteoprotegerin (OPG) and RANKL.Results After stastins was added,the level of animal and cells calcification decreased (In the animal model,the Ca2+ content,ALP activity,osteocalcin decreased respectively from (1.39 ± 0.04) mg/g,72.46±14.78,1.05±0.05 to (1.01 ±0.03) mg/g,48.39±9.27,0.56±0.04;and in the cell model from (120.66±7.63)μg/mgprotein,98.02 ± 9.56,0.95 ±0.03 to (95.57± 8.47) μg/mg protein,65.34 ± 8.16,0.65 ± 0.02 in the cell model,all P ＜ 0.05),and the ratio of OPG/RANKL increased obviously (all P ＜ 0.05).Conclusion Statins could inhibit the arterial calcification,the mechanism was probably connected with its function of increasing ration of OPG/RANKL.

Objective To study the effects of bone marrow mesenchymal stem cells(MSC)on the mineralization of calcified vascular smooth muscle cells (CSMC).Methods Osteogenic differentiation of vascular smooth muscle cells (SMC)was induced by osteogenic medium for 14 days.Then von Kossa staining was performed to observe the mineral deposits in SMC.The transwell system was used to establish the indirect co-culture environment.And hence,CSMC was indirectly cocultured without or with MSC at a ratio of 1:1 in the down plates or up plates.The cells were divided into 4 groups.Normal group included the normal SMC cultured with the normal medium.Calcification inducers group included the normal SMC cultured with the osteogenic medium.Calcified cells group included CSMC cultured with the normal medium.MSC intervention group included MSC and CSMC cultured with the normal medium.14 days later,SMC from 4 groups was harvested.Alkaline phosphatase(AKP)activity was detected to evaluate the severity of calcification.Moreover,the protein levels of Wnt5a and β-catenin were measured by Western blot analysis.Results Compared with normal group,the calcification inducers group showed that AKP activity,Wnt5a and β-catenin protein levels were notably increased.Additionally,AKP activity and the protein levels of Wnt5a,β-catenin were decreased in calcified cells group as compared with calcification inducers group.Furthermore,AKP activity and the protein levels of Wnt5a,β-catenin were significantly reduced(P＜0.01)in MSC intervention group versus calcified cells group Conclusions When MSC is indirectly co-cultured with CSMC,MSC can relieve the vascular calcification,which might be mediated by blocking the Wnt5a/β-catenin signaling through immunomodulatory and paracrine.

BACKGROUND:Our previous study showed that the combined Chinese herbs containing scutellaria baicalensis georgi and gardenia jasminoids ellis inhibited atherosclerosis. In this study, we sought to determine if baicalin and geniposide could inhibit atherosclerosis through Wnt1 and dickkopf-related protein-1 (DKK1). METHODS:The wild-type and ApoE-/- mice were treated with baicalin, geniposide, and baicalin plus geniposide daily by gavage for 12 weeks. Blood lipid levels were measured with an automatic biochemistry analyzer. Aortic atherosclerotic lesion areas were analyzed with Image-ProPlus software. The mRNA and protein expression of DKK1, Wnt1 and nuclear factor-κB (NF-κB) were measured with RT-PCR and Western Blot. Serum levels of interleukin-12 (IL-12) were quantified with ELISA. RESULTS:The baicalin or geniposide monotherapy as well as combination therapy inhibited the development of atherosclerotic lesions, increased Wnt1 and decreased DKK1 expression and elevated the ratio of Wnt1/DKK1 compared with high-lipid diet group. However, only baicalin or geniposide monotherapy decreased NF-κB expression. Moreover, baicalin and geniposide mono- or combination therapy lowered IL-12 levels. Geniposide reduced both serum total cholesterol and low density lipoprotein levels, while baicalin either alone or in combination with geniposide did not affect serum lipid levels. In human, umbilical vein endothelial cells stimulated by oxidized low density lipoprotein, baicalin and geniposide also increased Wnt1 and decreased DKK1 expression and elevated the ratio of Wnt1/DKK1. CONCLUSIONS:Baicalin and geniposide exert inflammation-regulatory effects and may prevent atherosclerotic lesions through enhancing Wnt1 and inhibiting DKK1 expression.

Vascular calcification (VC) is widely considered to be a crucial clinical indicator of cardiovascular disease. Recently, certain properties of mesenchymal stem cells (MSCs) have been hypothesized to have potential in treating cardiovascular diseases. However, their effect on the initiation and progression of VC remains controversial. The present study aimed to investigate whether MSCs indirectly mediate VC and their impact on the Wnt signaling pathways. A Transwell system was selected to establish the indirect co‑culture environment, and hence, vascular smooth muscle cells (VSMCs) were indirectly co‑cultured in the presence or absence of MSCs at a ratio of 1:1. Osteogenic medium (OS) was added to imitate a calcifying environment. Fourteen days later, VSMCs in the lower layers of the Transwell plates were harvested. Alkaline phosphatase activity and calcium nodules were markedly increased in calcific VSMCs induced by OS. However, these parameters were significantly decreased in VSMCs by indirectly co‑culturing with MSCs in the same medium. Furthermore, the messenger RNA expression levels of osteopontin and osteoprotegerin were notably increased in VSMCs cultured in OS, but reduced by indirect interaction with MSCs. In addition, the activities of canonical and noncanonical Wnt ligands, wingless‑type MMTV integration site family, number 5A (Wnt5a), receptor tyrosine kinase‑like orphan receptor 2 (Ror2) and β‑catenin, which are important in the process of VC, were downregulated by indirect contact with MSCs in OS. Thus, indirect co‑culture with MSCs inhibits VC and downregulates the Wnt signaling pathways.

目的：探讨破骨样细胞中骨保护素（ OPG）和 NF－κB配体受体（ RANKL）表达情况。方法单独培养骨髓单核细胞前体，用巨噬细胞集落刺激因子（ M－CSF）和维生素D诱导其转化为破骨样细胞，在0、5、10、15 d用光镜观察和TRAP染色（抗酒石酸染色）评估破骨样细胞的转化程度，用RT－PCR检测OPG和RANKL的表达情况；然后构建主动脉中膜平滑肌细胞（ SMC）与骨髓单核细胞前体共培养的模型，用维生素 C 和β－磷酸甘油诱导SMC转化为成骨样细胞，并在0、5、10、15 d用同样方法再次检测共培养中破骨样细胞转化的情况，以及两种细胞中OPG和 RANKL的表达情况。结果不管是单独培养、还是共培养，破骨样细胞中始终没有OPG和RANKL的表达。结论破骨样细胞的转化可能主要受成骨样细胞分泌的OPG和RANKL调控，本身并没有分泌OPG和RANKL进行自身调节的机制存在。

目的:探讨他汀类药物对动脉中膜钙化的抑制作用及其对骨保护素(OPG)/NF-κB配体的受体(RANKL)的影响.方法:构建大鼠的主动脉中膜平滑肌细胞(SMC)的钙化模型,随机分为钙化组、他汀组和对照组.钙化组使用β-磷酸甘油、维生素C对SMC来进行钙化诱导;他汀组在上述基础上加入阿托伐他汀.用Von Kossa染色,钙离子含量测定,碱性磷酸酶(ALP)活性检测,骨钙素的Western Blot检测来判定细胞钙化的程度,用实时定量PCR检测细胞钙化过程中伴随的OPG和RANKL的表达情况.结果:加入他汀类药物后使得细胞钙化的程度得到减轻,同时细胞表达的OPG/RANKL比值也得到明显上升(均P＜0.05).结论:他汀类药物具有抑制动脉钙化的作用,其机制可能与提高OPG/RANKL的比值有关.

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.

Hyperlipidemia is considered an independent risk factor for renal dysfunction and induces a significant increase in the expression of inflammatory mediators, which can be used to evaluate the degree of renal injury. Baicalin is widely used in traditional Chinese herbal medicine and has multiple pharmacological effects. The present study investigated whether baicalin can attenuate the expression of vascular cell adhesion molecule 1 (VCAM-1) via a reduction in the expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in the kidney of apolipoprotein E (ApoE)-knockout (KO) mice fed a high cholesterol diet. These mice were used as a model of atherosclerosis and were treated with baicalin (100 mg/kg/day) daily by gavage for a period of 12 weeks. By contrast, wild-type male C57BL/6J mice were fed a standard diet. Blood samples were obtained from the angular veins of the mice to measure the total cholesterol (TC) and the expression levels of VCAM-1, MCP-1 and IL-6 in the kidney tissues of the mice were analyzed using reverse transcription quantitative polymerase chain reaction and western blot analysis. Following oral administration of baicalin, no significant difference was observed in the TC in the baicalin group compared with the high cholesterol diet control group. The TC was significantly higher in the AopE-KO mice compared with the wild-type male C57BL/6J mice. The expression levels of VCAM-1, MCP-1 and IL-6 in the kidney tissues of the baicalin group were lower compared with those in the high cholesterol diet control group. The results suggested that baicalin decreased the expression levels of pro-inflammatory mediators and prevented kidney dysfunction in the ApoE-KO mice fed a high cholesterol diet.

Baicalin and geniposide, which are respectively isolated from Scutellariae radix and Gardenia jasminoides, have been known to exhibit a number of pharmacological effects, including anti-inflammatory and anti-oxidant. Here, we primarily aimed to observe the protective effects of these two Chinese herbs on inhibiting the development of atherosclerosis in apolipoprotein E knockout mice via lipids regulation and immunoregulation. After the ApoE−/− mice with high-cholesterol diet had received 12-weeks׳ oral administration of either baicalin or geniposide (100mg/kg), atherosclerotic plaque areas in aorta were measured and exhibited a prominent decrease in the treated mice. We then assayed serum lipids levels, serum Treg-cell-associated cytokines (TGF-β1 and IL-10) and the frequency of splenic Treg cells. We found that geniposide notably decreased serum TC and LDL-c. Both baicalin and geniposide treated mice showed much more splenic Treg cells and the correlated cytokines (TGF-β1 and IL-10). Foxp3, as the marker of Treg cell, was detected in atherosclerotic lesions, and we found that Foxp3 expression at both mRNA and protein levels was up-regulated in addition to increased Foxp3 positive Treg cells detected by immunohistochemistry in baicalin or geniposide treated mice. In conclusion, baicalin and geniposide up-regulated the expression of foxp3, promoted the number and function of Treg cells and ameliorated the atherosclerotic lesions progression partly through lipids regulation and immunoregulation.

Objective To study the differentiation of bone marrow mesenchymal stem cells in artery tissue under different calcification environments.Methods We made a vascular calcification model using warfarin,vitamin K1 and vitamin D3.After the model was successfully made,we took artery tissue of normal SD rat arteries and calcified arteries co-cultured with MSC,which were divided into three groups.The normal group included normal artery tissue with MSC; calcified inducers group included calcified inducers (dexamethasone with β-glycerophosphate and ascorbic acid),normal arterial tissue and MSC; calcification group included calcified artery tissue and MSC.Each group was cultured for 3 weeks.On the 10th day of the experiment,osteoprotegerin (OPG) protein secretion was detected by ELISA.After three weeks,changes of cell morphology were observed using inverted microscope,and total protein content and alkaline phosphatase (ALP) activity were detected by ELISA.In additional,the Ror2 (receptor tyrosine kinase-like orphan receptor 2) mRNA expression was detected by using RT-PCR method.Results MSC in calcification group spontaneously proliferated and differentiated to osteoblast-like cells.Compared with normal group,calcification group showed that the total protein content,ALP activity of bone metabolism markers,OPG were significantly elevated,while Ror2 mRNA expression was significantly decreased.MSCs in calcified inducers group did not differentiate to osteoblast-like cells,and the total protein content and OPG were increased,while ALP activity had no significant difference as compared with the normal group.However,Ror2 mRNA expression was lower in calcified inducers group than in normal group,while was higher than that in calcification group.Conclusions MSCs proliferate into bone-like differentiation in vascular calcification environment and aggravate the vascular calcification.And in normal vascular with calcified inducers environment,MSCs proliferate into smooth muscle cell differentiation and rehabilitate the vascular calcification.These phenomenons may be related to the Ror2 expression in artery.

Atherosclerosis is a systemic inflammatory disease characterized by the accumulation of dendritic cells (DCs) and other types of immune cells in atherosclerotic plaque. In this study, baicalin and geniposide were isolated from Scutellaria baicalensis Georgi and Gardenia jasminoids Ellis, which are the plants used in traditional Chinese medicine to treat a variety of inflammatory diseases. We then investigated whether baicalin and geniposide could induce regression of atherosclerotic lesions in ApoE-/- mice fed a high cholesterol diet and used as a model of atherosclerosis. Following model induction, these mice were treated with baicalin (100mg/kg), geniposide (100mg/kg), and then a mixture containing baicalin (100mg/kg) and geniposide (100mg/kg) administered daily by gavage for a period of 12weeks. The combined administration of baicalin and geniposide significantly reduced atherosclerotic lesions, and modulated the phenotype of dendritic cells in bone marrow and atherosclerotic plaque. Geniposide lowered both plasma lipid levels and DC numbers, while baicalin administered either alone or in combination with geniposide did not decrease plasma lipids. Our results suggest that baicalin and geniposide may have immune-regulatory effects and prevent the formation of atherosclerotic lesions by decreasing the DC numbers, and inhibit DC maturation in bone marrow and infiltration into lesions.