Effects of herbs of activation blood on atherosclerotic plaque morphology in ApoE gene-deficient mice

AIM: To observe the effects of six common traditional Chinese herbs of activating blood, paeoniae rubra radix, salviae miltiorrhizae radix, ligustici, rhizome, notoginseng radix, pruni persicae semen and wine staemed radix et rhizome, on atherosclerotic plaque structure and stabilization in ApoE gene-deficient mice. METHODS: Four sections of the aortic root were choosen and stained with hematoxylin and masson. All sections were measured with Image-Pro Plus Version 4.5.1 (IPP) software. RESULTS: Compared with the model group, plaque area corrected by cross-sectional vessel wall area reduced significantly in salviae miltirrhizae radix treatment group, lipid core area reduced in paeoniae rubra radix group, pruni persicae semen and wine steamed radix et rhizome treatment group, minimum thickness of fibrous cap became thicker significantly in salviae miltiorrhizae radix, ligustici, rhizome, pruni persicae semen and wine steamed radix et rhizome treatment group. CONCLUSION: These Chinese herbs may stabilize the atherosclerotic plaques in ApoE gene-deficient mice by interfering their structure, but their effects do not parallel with their activating blood efficacy in traditional Chinese medicine.     

Overexpression of SHP-1 mediated by lentivirus restrains atherosclerotic progression in mice

AIM: To investigate the role of SH2-domain-containing protein-tyrosine phosphatase-1 (SHP-1) lentivirus in atherosclerotic mice. METHODS: ApoE knock-out mice were randomly assigned to 3 groups: control group, GFP transfection group and SHP-1 transfection group. All mice were placed with carotid collars on the right common carotid arteries near its bifurcation, following feeding with high-fat diet for 8 weeks and then transfected with GFP blank vector or SHP-1 lentivirus (SHP-1-LV). The fluorescence density of the plaques, body weight, the levels of plasma total cholesterol (TC) and triglyceride (TG) were determined at 1st, 2nd, and 6th week after lentivirus transfection. Furthermore, the mRNA and protein expression of SHP-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP)-2 and MMP-9 were analyzed by real-time PCR and Western blot. Additionally, pathological analysis of the plaques was also performed by HE and oil red O staining. RESULTS: The fluorescence of the plaques was observed at 1st, 2nd, and 6th week after lentivirus transfection, with a highest density at 2nd week. The body weight and the levels of TC and TG in the mice were not influenced by lentivirus transfection. Moreover, SHP-1-LV transfection significantly upregulated the expression of SHP-1 at mRNA and protein levels, but inhibited the expression of IL-6, TNF-α, MMP-2 and MMP-9. In addition, SHP-1-LV transfection also decreased the plaque size ratio and lipid content in right common carotid arteries. CONCLUSION: SHP-1 overexpression accelerates the regression of atherosclerotic plaque, thus emerging SHP-1 as a target for prevention and treatment of atherosclerosis.     

Effects of several herbal extractives with the effect of promoting blood flow and detoxication on atherosclerotic plaque stability in aorta of ApoE-gene knockout mice

AIM: To observe the effects of saponins of panax pseudo-ginseng, coptis chinensis extractive, giant knotweed rhizome and rhubarb extractives on the stability of vulnerable atherosclerotic plaques in the aortic roots of fat-fed ApoE knockout mice. METHODS: Eighty ApoE knockout mice were fed high-fat diet for 26 weeks. Thereafter, they were randomized to one of seven groups and treated with these herbal extractives for 13 weeks. The morphology and composition of atherosclerotic plaques in aortic roots were examined in tissue sections. Four sections were chosen and stained with Movat and HE staining, respectively, and the protein expressions of smooth muscle in plaque were determined by immunohistochemistry. The percentages of the plaque composition of foam cells, extracellular lipids, smooth muscle cells and collagen in each area were counted in the whole plaque. The number of buried caps and the ratio of the length of fibrous cap/plaque surface were also counted. The effect of the drugs on plaque stability was evaluated by using the vulnerability index 〔(foam cells＋extracellular lipids)/(collagens+smooth muscle cells)〕 and the number of buried fibrous caps. RESULTS: The vulnerability indexes in all drug treatment groups were decreased (P<0.05, P<0.01). The giant knotweed rhizome and rhubarb extractives, which had both the effect of promoting blood flow and detoxication, inhibited it the most significantly among them (P<0.01). The significant differences between the promoting blood flow group (saponins of panax pseudo-ginseng) and detoxication group (coptis chinensis extractive) were observed. Furthermore, the mean number of buried caps in plaque of giant knotweed rhizome extractive group and coptis chinensis extractive group were significantly decreased compared with that of control(P<0.01, P<0.05). CONCLUSION: Saponins of panax pseudo-ginseng, coptis chinensis extractive, giant knotweed rhizome and rhubarb extractives stabilize vulnerable plaque by changing plaque composition in clinical commended doses. Among them the herbal extractives with both the effects of promoting blood flow and detoxication (giant knotweed rhizome and rhubarb extractives) have the best effect on stabilizing vulnerable atherosclerotic plaque. Our investigation indicates that this kind of herbal extractives may be have similar effectiveness on stabilizing vulnerable atherosclerotic plaque for decreasing acute cardiovascular events, which is superior to herbs that has the effect of promoting blood flow only or detoxication only.     

Effect of zhiganning on the expressions of HSP60 and HSP70 in the hepatocytes of rats undergoing steatohepatitis

AIM: To observe the expressions of HPS60 and HPS70 in hepatocytes in rats under treatment with zhiganning on steatohepatitis. METHODS: Male SD rats were randomly divided into large dose zhiganning group, small dose zhiganning group, ursodeoxycholic acid (UDCA) group, model group, normal control group. Except the normal control group, all the other rats were fed with high fat (88% standard diet, 10% lard, 2% cholesterol) and 35% alcohol 10 mL/kg twice a day. Prophylactic drugs were used at the same time. All rats were sacrificed at the 9th week. Routine histologic features of hepatic sections were observed by HE staining and penetrated electron microscope. The expressions of HSP60 and HSP70 in the liver were detected by immunohistochemistry, respectively. RESULTS: ⑴ The degree of steatohepatitis in the large dose zhiganning group and ursodeoxycholic acid (UDCA) group were significantly decreased compared with that in model group (P<0.05). ⑵ The expression of HSP70 in the large dose zhiganning group and ursodeoxycholic acid (UDCA) group were significantly higher than that in either model group or normal control group (P<0.05, P<0.01, respectively). The expression of HSP60 in the large dose zhiganning group was significantly higher than that in either model group or normal control group (P<0.05, P<0.01, respectively). ⑶ In the large dose zhiganning group and ursodeoxycholic acid group, ultramicroscopic structure of liver was nearly normal, which was significantly improved compared with model group. CONCLUSION: The results indicate that zhiganning and UDCA effectively prevente the steatohepatitis in rats induced by high fat diet and alcohol. The enhanced expressions of HSP60 and HSP70 may play an important role in the prevention of liver from injury.     

Effects of CpG ODN on dendritic cells in anti-hepatocarcinoma action

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL.     

Effects of dendritic cells co-cultured with CIK cells on renal carcinoma cells

AIM: To study the effects of CIK cocultured with DC that pulsed with RCC antigen on renal carcinoma cells.METHODS: DC and CIK cells were generated respectively by cytokines from PBMC of healthy blood donor.Cell surface markers were analyzed by flow cytometry.Then CIK were cocultured with autologous DC that was (or not) pulsed with RCC antigen (786-0 cells).Cytotoxic activity against 786-0 or PC3 cells was measured by MTT assay under three different conditions: CIK cocultured with DC which was pulsed with 786-0 antigen (group A);CIK cocultured with DC which is not pulsed with 786-0 antigen (group B);CIK without DC (group C).RESULTS: The cytotoxic activity of three groups against 786-0 cells was (70.64±8.26)%, (53.40±7.33)%, (46.64±6.01)%, respectively (E/T=20∶〖KG-*2〗1).Significant differences between group A and group B or between group A and group C were observed (P<0.05).There was a significant difference in cytotoxic effects of group A against 786-0 and PC3 cells (P<0.05).CONCLUSION: Coculture of CIK with autologous tumor-pulsed DC leads to a significant increase in cytotoxic activity against renal carcinoma cells, and cytotoxicity mediated by RCC lysate antigen possess stronger specificity.     

Effects of iNKT cells from OVA-induced asthmatic mice combined with OVA on phenotype and function of bone marrow-derived dendritic cells in vitro

AIM:To investigate the effects of invariant natural killer T-cells (iNKT cells) from ovalbumin (OVA)-induced asthmatic mice combined with OVA on the phenotypic and functional characteristics of bone marrow-derived dendritic cells (BMDCs) in vitro. METHODS:The BMDCs from wild-type (WT) BLAB/c mice were co-cultured with purified iNKT cells from WT mice immunized and challenged with OVA in the presence of 100 mg/L OVA (iNKT cells plus OVA group) or PBS (iNKT cells plus PBS group) for 20 h, and were also cultured with 50 mg/L LPS (LPS group), 100 mg/L OVA (OVA group) or PBS (PBS group) for 20 h. The expression of MHC-Ⅱ, CD40, CD86, and CD80 on the BMDCs was measured by flow cytometric analysis, and the levels of interleukin-12 (IL-12) p70, IL-6, tumor necrosis factor-α (TNF-α) and IL-10 in the culture supernatant were measured by ELISA. Splenic CD4⁺ T cells from DO11.10 transgenic mice were co-cultured for 48 h with the above mentioned BMDCs, and then the concentrations of IL-4 and interferon-γ (IFN-γ) in culture supernatants were measured by ELISA. RESULTS:The expression of MHC-Ⅱ, CD80, CD86 and CD40, and releases of proinflammatory cytokines by BMDCs in iNKT cells plus OVA group were comparable to those in the LPS group (P>0.05), but significantly higher than those in iNKT cells plus PBS group, OVA group, and PBS group (P<0.05 or P<0.01). The concentration of IL-4 in culture supernatants from BMDCs in iNKT cells plus OVA group co-cultured with DO11.10 CD4⁺ T cells was similar to that in LPS group (P>0.05), but markedly higher than that in iNKT cells plus PBS group, OVA group, and PBS group (P<0.01). CONCLUSION:Bone marrow-derived dendritic cells undergo immunogenic maturation upon interaction with iNKT cells in the presence of OVA.     

Effects of CpG ODN on dendritic cells and its mechanisms

Oligodeoxynucleotide containing unmethylated cytosine phosphate-guanosine motif(CpG ODN) may induce high expression of CD80, CD86, CD83, HLA I and HLAⅡ molecules on dendritic cells(DC) and stimulate DC to produce high level of IL-6, IL-12, TNF-α and IFN-α. CpG ODN is demonstrated in vivo to be a very potent adjuvant for Th1 cells, regulating Th0 cells to develop toward Th1 cells. Its role for DC is characteristics of CpG ODN sequence specificity and species specificity. CpG ODN is, at present, considered as a pathogen associated molecular pattern which binds its specific receptor，Toll-like receptor 9，then functions through TLR/IL-1R signaling pathway. It may represent a new therapeutic drug for broad applications in infectious disease, autoimmune disease, allergy and cancer therapy.     

Effects of uric acid sodium salt on antibody response,dendritic cells and delayed-type hypersensitivity in BALB/c mice

AIM: To investigate the effects of uric acid sodium salt (UANa) as adjuvant on humoral and cellular immune response in BALB/c mice. METHODS: BALB/c mice were immunized with trichosanthin (TCS) as antigen together with UANa suspension as adjuvant. The antibody titers of IgG were detected by enzyme-linked immunosorbent assay. Dendritic cells (DC) were induced in vitro, the phenotypes of DC were analyzed by flow cytometry and the effect of UANa on DC maturity was evaluated. A delayed-type hypersensitivity (DTH) model was used to analyze the effect of UANa on cellular immune responses in vivo. The in vitro proliferation of lymphocytes was determined by ConA stimulation. RESULTS: Freunds adjuvant greatly enhanced the antibody response of mice to TCS, while UANa adjuvant failed to promote the antibody response but significantly reduced the antibody response as compared to TCS only. No effect of UANa on the expression of CD11c and CD83 in DC was observed by flow cytometry analysis. However, UANa significantly enhanced the expression of MHC II molecule. In the DTH model, UANa enhanced the degree of allergen-induced ear swelling and promoted the ability of lymphocyte proliferation in vitro. CONCLUSION: UANa suspension as adjuvant significantly enhances the cellular immune response but inhibits the humoral immune response to a certain degree, suggesting that UANa has potential application in the vaccine research.     

Effect of adriamycin combined with sensitized dendritic cells on cervical tumor-bearing mice

AIM：To explore the immunotherapeutic effect of adriamycin (ADM) combined with frozen-thawed antigen-sensitized dendritic cells (DCs) on cervical tumor-bearing mice. METHODS：The U14 cervical cancer model of Kunming mice was established by subcutaneous implantion of U14 cells in axillary fossa. DCs vaccine was prepared by U14 cervical cancer cell frozen-thawed antigen-sensitized mouse bone marrow-derived DCs. Mature phenotype of sensitized DCs was identified by flow cytometry. Tumor-bearing mice were randomly divided into 4 groups and treated for 3 cycles with PBS (control), DCs vaccine, ADM and ADM combined with DCs vaccine, respectively. The tumor volume was evaluated. The tumor weight and the levels of interleukin-2 (IL-2), IL-12 and interferon γ (IFN-γ) in the serum were determined by ELISA on the 21st day. RESULTS：Cancer cell frozen-thawed antigen-sensitized DCs had higher expression levels of CD11C, CD80 and CD86. The volume and weight of the tumor in ADM combined with DCs vaccine group were less than those in ADM group, DCs vaccine group and control group. The tumor inhibitory rate in combination group was higher than that in the other 3 groups. Compared with the other 3 groups, the serum levels of IL-2, IL-12 and IFN-γ in combination group significantly increased. CONCLUSION：ADM combined with tumor antigen-sensitized DCs vaccine can strengthen the animal antitumor immune response and effectively inhibit the growth of tumor in cervical tumor-bearing mice.     

Significance of soluble CD40 ligand in the vulnerability of coronary atherosclerotic plaque

AIM: To evaluate the significance of serum soluble CD40 ligand (sCD40L) in the vulnerability of coronary atherosclerotic plaque, the relationship between the level of sCD40L and the stenosis degree of the coronary artery by the coronary angiography (CAG), and other inflammatory factors. METHODS: According to WHO diagnostic criterior of coronary heart disease (CHD) and the results of CAG, 84 cases of CHD and 20 cases of non-CHD (NCHD) were included in this study. 84 cases of CHD were divided into three groups: 30 cases in acute myocardial infarction (AMI) group, 30 cases in unstable angina (UA), 24 cases in stable angina (SA). The sera levels of sCD40L in four groups were detected by the enzyme-linked immunosorbent assay (ELISA) and the results were expressed with μg/L. CAG were all conducted in four cases and the results were further evaluated by Jenkins score. ESR and CRP were detected at the same time. RESULTS: The sera levels of sCD40L in four groups were significantly different (P<0.01). The level of sCD40L in AMI group (8.48±4.13) μg/L was higher than that in SA group (4.36±2.68) μg/L, P<0.01 and NCHD group (4.12±1.96) μg/L, P<0.01. The level of sCD40L in UA group (8.72±4.26) μg/L was higher than that in SA group and NCHD group (P<0.01). The level of sCD40L in UA group was slightly higher than that in AMI group, but the difference of two group is not significant (P>0.05). The level of sCD40L in SA group was slightly higher than that in NCHD group, but the difference of two group is not significant (P>0.05). The sera levels of sCD40L in CHD were significantly and positively correlated with Jenkins score (r=0.524, P<0.01). The sera level of sCD40L was positively correlated with the levels of CRP and ESR. CONCLUSION: The sera levels of sCD40L in the patients with various types of CHD are significantly different. The level of sCD40L in the patients with AMI and UA are significantly higher than those in SA and NCHD groups, which may reflect the vulnerability of coronary atherosclerotic plaque. The sera levels of sCD40L is increased with the increasing number of diseased coronary branches and Jenkins score, suggesting that sCD40L promotes atherosclerosis and also can be used as a parameter to predict pathological severity of coronary atherosclerosis. The level of sCD40L is obviously correlated with the levels of CRP and ESR.     

Effects of alkyl-lysophospholipids on proliferation and differentiation in HL-60cells

AIM: To explore the effect of alkyl-lysophospholipids (ALP) on the proliferation, apoptosis and differentiation of HL-60cells. METHODS: Proliferative potential was measured by colony formation assays. Apoptotic cells were detected by morphology, DNA gel electrophoresis and flow cytometry analysis. Both morphological criteria and NBT dye reduction were utilized to determine the extent of differentiation. RESULTS: After 9 h of incubation wit15 mg/L of ALP, apoptotic cells, identified by condensened and fragmented nuclei, were present. After 6 days of incubation wit1 mg/L of ALP, the NBT reduction rate in HL-60cells increased to 84.2±2.6%. CONCLUSION: ALP can induce apoptosis and differentiation, and inhibit growth in HL-60cells.     

Recovery of endothelial dysfunction with tolerogenic dendritic cell loaded with heat shock protein 60 in apolipoprotein E-null mice

AIM: To examine whether tolerogenic dendritic cells (DC) loaded with heat shock protein 60 (HSP60) could restore endothelial function in hypercholesterolemic apolipoprotein E (apoE)-null mice.METHODS: Bone marrow derived DC of the mice was loaded with HSP60 and co-cultured with rapamycin to generate tolerogenic DC.The tolerogenic DC, DC loaded only with HSP60 (DChsp) and saline were injected into the apoE-null mice at 6 weeks of age for two times at a one-week interval.C57BL/6 mice at the same age were taken as normal control two weeks after the last injection.Aorta was harvested for ex vivo vascular ring tension test.Immune parameters were also analyzed in vitro and in vivo.RESULTS: Compared with the non loaded DC, HSP60 pulsed DC expressed higher levels of CD86, and stimulated T lymphocytes to proliferation significantly, while the tolerogenic DC expressed lower levels of CD86, and inhibited T lymphocytes to proliferation.After immunization with different injection, Ach-induced relaxation was reduced significantly in DChsp group compared with saline group (P<0.01).Treatment of mice with tolerogenic DC restored endothelium-dependent dilation in a dose-dependent manner (P<0.01).The improvement in endothelial function was associated with a reduction in T cell response to HSP60.CONCLUSION: Our results indicate a rapid improvement in endothelial function with HSP60 tolerogenic DC immunization, and suggest that this immune therapy has significant vasculoprotective effects.     

Study on the atherosclerotic lesions in coronary artery of apoE gene knockout mice

AIM: To study the distribution and composition of the atherosclerotic lesions in the coronary artery of apoE gene knockout (-/-) mice, and to search for the mechanism of its occurrence and development. METHODS: The successive sections of the hearts of apoE-/- mice were made. All the coronary trunk and branches in myocardium from the opening of coronary artery leaving aorta were traced continuously by Movat staining. The length of the lesions and the distance from the beginning of the coronary was calculated depending on the numbers of sections. The caliber of the vessels was measured with computer and the components of the lesions was also observed by Movat staining. RESULTS: There were two kinds of lesions in apoE-/- mice, extending lesions which directly extended from the beginning of coronary artery and in situ lesions which formed at the branches of the coronary arteries. The in situ lesions tended to locate at the papillary muscles and near the bifurcation of branches in left ventricle. More large lesions in apoE-/- mice of 112 weeks than those of 60 weeks (P＜0.05) were observed. Infiltration of inflammatory cells in the adventitia was usually ahead of the two sorts of atherosclerotic lesions. The component of proteoglycan was decreased and the component of lipid was increased with the enlargement of the in situ lesions. Some large lesions may obstruct the small vessels. CONCLUSIONS: There are two kinds of atherosclerotic lesions (extending lesion and in situ lesion) within the coronary artery of apoE-/- mice. Both kinds of lesions are gradually increased with age in size. The infiltration of inflammatory cells in adventitia and the hemodynamic oppression by myocardial contraction are related to the occurrence and development of these lesions.     

Effects of progesterone on the maturation and immunologic function of dendritic cells from human peripheral blood

AIM: To study the effects of progesterone (P₄) on the maturation and immunologic function of dendritic cells (DCs) from human peripheral blood. METHODS: Cultured DCs were treated with P₄ at doses of 10-7 mol/L and 10-6 mol/L. The morphologic changes were observed under the scanning electronic microscope. The immunophenotypes of DCs in control and treated groups were analyzed by flow cytometry. IL-10 and IL-12 production in culture supernatant was examined by ELISA assay. The capability of the stimulatory activity of the DCs on allogeneic T cells in mixed reaction was tested by incorporation of ［3H］-TdR. RESULTS: Compared with control group, cultured DCs in the presence of P₄ displayed less dendritic pseudopod, expressed low levels of MHC-II, CD40, CD80 and CD86, and exhibited weakly activity in stimulating the proliferation of allogeneic T cells. Increase in IL-10 production and decrease in IL-12 production were observed. CONCLUSION: P₄ exerts negative effect on the maturation and immunologic function in dendritic cells from human peripheral blood.     

Experimental study on induction of atherosclerotic plaque instability in rabbits after transfer of wild-type p53 gene

AIM: To study the apoptotic role of wild-type p53 in induction of plaque instability in atherosclerotic rabbits. METHODS: Fifty-four New Zealand White rabbits underwent balloon-induced abdominal aortic wall injury and then were fed on a diet of 1% cholesterol. At the end of the eighth week, the rabbits were randomly divided into two groups: group A and group B. Recombinant adenovirus carrying p53 and β-galactosidase (LacZ) genes were injected in group A and B, respectively. Two weeks later, 10 rabbits each in group A and B was killed and the remaining rabbits all underwent pharmacological triggering with injection of Chinese Russell's viper venom and histamine. RESULTS: Compared with group B, p53 gene over-expression in group A resulted in a marked increase in number of positive apoptotic cells (2.5%±0.8% vs 1.0%±0.3%, P<0.05) and a significant decrease in vascular smooth muscle cells (47.5%±6.8% vs 80.4%±10.6%, P<0.01), the thickness of the fibrous cap ［(132.9±56.7）μm vs （181.8±59.7） μm, P<0.05］ and the cap/intima-media ratio (0.20±0.18 vs 0.21±0.11, P<0.05). CONCLUSION: Transfer of human wild-type p53 genes effectively promotes apoptosis of VSMCs in atherosclerotic plaques, which makes the plaques vulnerable to rupture.