The crude components of XMJ were purchased from Beijing Tong Ren Tang Chinese Medicine Co. Ltd. (Beijing, China). Lovastatin, H₂O₂, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and phenazine methosulfate (PMS) were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Zhibituo was obtained from Chengdu Diao Pharmaceutical Group Co., Ltd. (Chengdu, China). The RPMI-1640 culture medium and fetal calf serum were acquired from Gibco (Carlsbad, CA, USA). Wright-Giemsa (R-G) stain was purchased from Changsha Li Xin Biotechnology Co. (Changsha, China). IL-1, IL-6, ICAM-1, VCAM-1, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase (TIMP-2) and nuclear factor (NF)-κB were purchased from R&D Systems (Minneapolis, MN, USA). All other reagents were analytically pure and purchased in China.

Human aortic smooth muscle cells (HASMCs) were obtained from HASMC cell lines purchased from American Type Culture Collection (Manassas, VA, USA). The cells were routinely maintained in phenol red containing Dulbecco’s modified Eagle’s medium supplemented with 15% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml phytomycin in a 37°C incubator containing 5% CO₂. The third generation of HASMCs was used in the study. The cells were randomly divided into eight groups and incubated with the corresponding drugs for 24 h. The cells in the first group were incubated with Kreb’s solution and were assigned to the blank control group (n=6). The cells in the second group were incubated with 500 mg/l XMJ and were assigned to the XMJ control group (n=6). The cells in the third group were incubated with 200 μmol/l H₂O₂ and were assigned to the model group (n=6). The cells in the fourth group were incubated with 1 μmol/l lovastatin and 200 μmol/l H₂O₂ and were assigned to the lovastatin group (n=6). The cells in the fifth group were incubated with 50 μmol/l zhibituo and 200 μmol/l H₂O₂ and were assigned to the zhibituo group (n=6). The cells in the sixth group were incubated with 25 μmol/l XMJ and 200 μmol/l H₂O₂ and were assigned to the low-dose XMJ group (n=6). The cells in the seventh group were incubated with 50 μmol/l XMJ and 200 μmol/l H₂O₂ and were assigned to the middle-dose XMJ group (n=6). The cells in the eighth group were incubated with 100 μmol/l XMJ and 200 μmol/l H₂O₂ and were assigned to the high-dose XMJ group (n=6). The cultured cells were collected after each treatment for subsequent tests.

Approximately 1,000 g of the crude components of XMJ were placed in a container containing 6,000 ml water and heated for 1 h in a microwave oven at 1,000 W. The decoction of XMJ was obtained after filtering the extract through four gauzes. Thereafter, 6,000 ml water was added to the container and the above procedure was repeated. After mixing the former decoction of XMJ with the latter one, the mixture was filtered using sterile absorbent cotton. XMJ was refined by ultra-filtration technology with water decoction at a pressure of 0.5 kPa/m³, a temperature of 25°C, and a flow rate of 100 l/h/m². Approximately 5,000 ml of filtrate was then condensed to 1,000 ml, which was equivalent to 1 g of XMJ/ml of liquid medicine. Finally, the refined liquid was labeled and stored in a refrigerator at 4°C.

Seed cells were placed into 96-well plates (100 μl/well) and cultured in RPMI-1640. Thereafter, the null medium was discarded and replaced with 100 μl fresh medium and 25 μl XTT and PMS mixture. After cell culture for 4 h, the optical density (OD) was measured at 450 and 630 nm wavelengths using an enzyme-linked immunosorbent assay (ELISA) plate reader (BioTek Instruments, Inc., Winooski, VT, USA). Each group contained six wells. The result was obtained from the average value of these six wells.

The cells were plated in a 24-well plate at 5×10⁴ cells/ml, treated with XMJ (25, 50 and 100 μmol/l), lovastatin and zhibituo, respectively, for 24 h. The sterile slides were placed in a 24-well plate in advance for the adherence of cells. The slides were then taken out and washed twice with phosphate-buffered saline (PBS). After air-drying at room temperature, Wright-Giemsa dye was added to rapidly cover the slide. The same amount of PBS was added a few minutes later. The Wright-Giemsa dye and the PBS were mixed using a rubber pipette bulb. The dye was then rinsed with water, dried and sealed, and the cells were examined under a microscope (Olympus, Fukushima, Japan).

Seed cells were placed into 6-well plates in 2 ml concentrations/well. To detect the biochemical indicator of the cell supernatant fluid, the culture fluid was collected to measure the superoxide dismutase (SOD) and malondialdehyde (MDA) activity, following the manufacturer’s instructions. The reagents and kit used to measure SOD and MDA were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China).

Seed cells were placed into 6-well plates (2 ml/well). The supernatant fluid of the various wells in the plates was collected by an ELISA reagent kit, according to the manufacturer’s instructions (R&D Systems Inc., Minneapolis, MN, USA). The OD value of each well was measured at a 450 nm wavelength. The OD value was assigned as the abscissa, while the standard liquid concentration of the reagent was assigned as the ordinate. The relevant curve was drawn and the curve equation was calculated. The OD values of the samples were substituted into the equation of the standard curve, and the IL-1, IL-6, ICAM-1, VCAM-1, MMP-2, TIMP-2 and NF-κB values were calculated.

Seed cells were placed into 24-well plates (1 ml/well). After 24 h of cell culture, a line of the same width was drawn in each well with a 200 μl pipette tip, and the plates were washed three times with sterile PBS. The cells were cultured in RPMI-1640 medium without serum, and exposed to XMF (25, 50 and 100 μmol/l), lovastatin and zhibituo, respectively, for 24 h. The entire process was recorded. The scratch width was detected using scratch image software 3.22 (Olympus). Six scratch belt widths were collected in each well, and the average value of the widths was calculated and compared.

All data are shown as the mean ± standard error. Single factor variance and Student-Newman-Keuls multiple comparison analyses were used to compare data from different groups. Comparisons were performed using statistical software SPSS 13.0 (SPSS, Inc., Chicago, IL, USA. P<0.05 was considered to indicate a statistically significant result.

The HASMCs in the control group were arranged closely with abundant cytoplasms and intact cell membranes. The HASMCs demonstrated typical ‘peak-valley’-like growth. The number of cells in the normal group was lower than that in the model group. The normal group had loose cell connections and exhibited cytoplasm shrinkage, whereas the model group lost its typical growing appearance but had a fusiform cell presentation. The drugs inhibited cell proliferation to varying degrees. Certain damaged cells were restored with plumper cytoplasms and clearer profiles. However, these effects were much clearer in the high-dose XMJ group. Certain cells in the high-dose XMJ group reverted to a near-normal state. However, no significant effects were observed in the zhibituo and lovastatin groups. The cell shrinkage in the lovastatin group was particularly evident (Fig. 1).

Cells that proliferate after HASMC treatment are damaged and gradually migrated to the vascular intima from the tunica media vasorum, thus inducing plaque formation (17). Excessive proliferation of HASMCs is also an important factor in high blood pressure (18). The results showed that HASMCs in the model group were significantly more proliferative after 24 h of interaction than the cells in the normal group (Fig. 2, P<0.05). The results showed that HASMCs in the model group were significantly more proliferative after 24 h of interaction than the cells in the normal group, proliferative HASMCs in the middle-dose XMJ group, low-dose XMJ group and high-dose XMJ group were significantly inhibited after 24 h of interaction in a dose-dependent manner.

Inflammation induces atherosclerotic plaque formation and occurs during the development of AS. Therefore, we examined the inflammatory factors MMP-2 and TIMP-2 in the model group. The results showed that the levels of both factors were significantly decreased in the model group compared with those in the normal group (P<0.05); however, the levels of both factors increased significantly after treatment with different concentrations of XMJ compared with those in the model group (P<0.05). The most significant effect was observed in the middle-dose XMJ group. The levels of both factors also increased in the zhibituo and lovastatin groups; however, the effect was not significant compared with those in the XMJ groups (Table I).

The activation mechanism of the NF-κB pathway and its effect on related inflammatory factor content (including IL-1 and IL-6 levels; Fig. 3) were investigated. The results showed that the NF-κB content in the model group was significantly increased compared with that in the normal group (P<0.05), thus indicating that the NF-κB pathway was significantly inhibited. However, the NF-κB content decreased significantly after treatment with different concentrations of XMJ compared with those in the model group (P<0.05). This result indicates that inhibition of NF-κB pathway activation by XMJ reduced the inflammatory response. The NF-κB content decreased significantly in the zhibituo group compared with that in the model group (P<0.05). No significant difference was observed between the zhibituo and XMJ groups. The NF-κB content also decreased in the lovastatin group compared with that in the model group (P<0.05). However, the effect of the lovastatin group was greater than that of the XMJ and zhibituo groups (Table I).

Lipid peroxidation induces plaque formation during AS development (19). Plaque formation may be controlled if lipid peroxidation is inhibited effectively. SOD is a well-known and important antioxidant enzyme. Therefore, we first detected the SOD activity (Fig. 4). The SOD activity increased significantly in a concentration-dependent manner after treatment with various concentrations of XMJ. The SOD activity was significantly different in the high-dose XMJ group from that in the model group (P<0.05). This result showed that XMJ may effectively promote the synthesis of antioxidant molecules such as SOD by HASMCs, thus effectively inhibiting the occurrence of oxidation. The MDA content, which is an important indicator of oxidative stress, was also measured. The MDA content in the model group increased significantly compared with that in the normal group, but decreased significantly after treatment with various concentrations of XMJ (P<0.05). The MDA content in the zhibituo and XMJ groups was significantly decreased compared with that in the model group (P<0.05), thus indicating that XMJ exerts antioxidant effects by inhibiting MDA synthesis.

The proliferation and adhesion capacity of HASMCs from the tunica media to the tunica intima is enhanced in injured HASMCs (20). Therefore, the expression of adhesion factors ICAM-1 and VCAM-1 was detected (Fig. 5). The results showed that the levels of the two adhesion factors were significantly increased in the model group compared with those in the normal group (P<0.05), but decreased significantly in a concentration-dependent manner after treatment with different concentrations of XMJ compared with those in the model group (P<0.05). The ICAM-1 content in the zhibituo and lovastatin groups decreased significantly (P<0.05) as did the VCAM-1 content. These results showed that XMJ reduced the adhesion and migration of HASMCs from the tunica media to the tunica intima. The scratch experiment showed that the scratch width narrowed significantly in the model group, and that the cell migration ability became stronger (Fig. 6). The scratch width widened significantly after treatment with different concentrations of XMJ. Cells were shed from the cell wall due to the reduction in cell adhesion. No significant difference was observed between the scratch distances in the zhibituo and lovastatin groups compared with those in the model group (Fig. 7).