Supported by Project of Shaanxi Administration of Traditional Chinese Medicine (zy06); Special Project of Education Department of Shaanxi Provincial Government (12JK1016); Program of Shaanxi Provincial Department of Science and Technology (2013jk4023).

1 Introduction

Liver disease is a common clinical disease, and hepatocellular injury is a common pathological basis for various types of liver diseases. Prevention and improvement of liver cell damage are major measures of liver disease treatment. RADIX BUPLEURI (Bupleurumchinense DC.) and its related drugs have an excellent effect on the treatment of various liver diseases. Toll-like receptor 4 (Toll-like receptor 4, TLR4) is an important recognition receptor that is located on the cell surface to identify pathogenic molecules. It can induce the occurrence of a variety of diseases such as inflammation, the NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a downstream TLR4 factor, TLR4 is activated it downstream NFκB activation can be enhanced, and finally leads to the release of a variety of inflammatory mediators in great quantities, produces various physiological effects, and causes damage to the liver. TLR4-NFκB signaling pathway is closely related to the occurrence and development of liver diseases, and has a certain relationship with the formation of liver fibrosis[1]. The previous study of our team has shown that the compatibility of Saikosapon d and Baicalin can reduce the liver fibrosis injury of rats[2], the mechanism of which may be connected with the regulation of TLR4-NFκB signaling pathway, but its effective component is still not clear. In this invitro experiment, the protective effects of Saikosaponon d and Baicalin on the CCl4 injured L-02 cells were observed, and the possible mechanism was discussed from TLR4-NFκB signaling pathway[3-4].

2 Materials

2.1 Cell lines Experimental liver cell lines: L-02 embryo liver cells, provided by the laboratory of Shaanxi University of Chinese Medicine, cultured in the improved RPMI-1640 medium containing 10% fetal bovine serum, under conditions of 37℃ and 5% CO2 and saturated humidity, subcultured every 2 to 3 days, and cells in logarithmic phase were taken for experiment.

2.2 Reagents Improved RPMI-1640 medium (Thermo Fisher Scientific Co., Ltd.), fetal bovine serum, trypsin (both bought from Beijing TransGen Biotech Co., Ltd.), SABC rabbit immunohistochemical (IGC) kit, NFκBp65, TLR4, DAB chromogenic reagent (both bought from Wuhan Boster Biological Technology Co., Ltd.), DMSO (Sigma-Aldrich), methylthiazolyl tetrazolium (MTT) assay (Amresco), alanine aminotransferase (ALT) assay kit, aspertate aminotransferase (AST) assay kit (bought from Changchun Huili Biotech Co., Ltd.).

Mike：中等强度的黑色水果味，酸度和酒精感都比较突出，酒体相对强劲，入口有浓郁的黑果和橡木桶带来的草本、香料味道。有氧化气息，略显老态。可以搭配香草汁牛扒/羊扒，烧鹅。

3.3 Effects of Saikosaponon d and Baicalin on AST and ALT levels of CCl4 injured L-02 cell culture medium L-02 cells in logarithmic growth phase were inoculated with 1640 culture medium containing 10% foetal bovine serum in 96-well culture plate with cell concentration of 1×105 pcs/mL, 100 μL per well, placed in CO2 incubator for 12 h, CCl4 intervention for 6 h, added drugs for acting 24 h, collected the supernatant (the blank group, CCl4 intervention group, 1.75 μg/mL group, 1.5 μg/mL group, and 1.25 μg/mL Saikosaponon d and Baicalin group). In accordance with the instructions of the assay kit, the serum aminotransferase ALT and AST absorbance (OD value) were read by microplate reader.

本试验过程中，小鼠各肠段肠黏膜SIgA呈现动态变化趋势，前3周逐渐上升，但在第4周的时候出现了全群下降，之后又恢复上升趋势，说明小鼠的免疫能力随着年龄的增长逐渐发展和完善，第4周的全群变化可能与气候突变导致应激所致。

3.2 Protective effects of Saikosaponon d and Baicalin on the CCl4 injured L-02 cells First, 8 mmol/L CCl4 was used (small volume of DMSO was used to promote dissolution, and final concentration of DMSO was lower than 0.1%)[6]. L-02 cells with the concentration of 1×105 pcs/mL were incubated in 96-well plate, 100 μL per well. After incubation for 12 h in CO2 incubator, removed the supernatant, added 100 μL of CCl4 for 6 h and removed the supernatant, added 100 μL of Saikosaponon d and Baicalin with concentration of 2.5, 2.25, 2.0, 1.75, 1.5 and 1.25 μg/mL, 24 h later, added 20 μL MTT to culture 4 h, and added 150 μL DMSO, shaken up on a micro-oscillator, and detected with Microplate Reader at 490 nm.

3 Methods

笙主要由笙苗（许多长短不一的竹管）、笙簧（装在竹管下端的铜制簧片）、笙斗（连接竹管和吹口的底座）三部分构成。吹奏时，用手指按着竹管下端所开的孔，使簧片与竹管中的气柱发生共鸣而发出乐音。

2.4 Experimental medicine According to the references[5-6] and pre-experimental results, Saikosaponon d and Baicalin were dissolved in DMSO at 1∶15 ratio.

2.3 Instruments Super Clean Workbench (SW-CJ-2FD Double-Person Single-Side Clean Workbench, Suzhou Purification Equipment Co., Ltd.); Cell Centrifuge (Zenith Lab (Jiangsu) Co., Ltd.); Inverted Microscope (Shanghai Optical Instrument Factory); Electronic Analytical Balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); CO2 Incubator (Shanghai Boxun Industrial Co., Ltd.); DG5033A Microplate Reader (Nanjing Huadong Electronics Group Medical Equipment Co., Ltd.).

应用HITACHI HI VISON Preirus彩色多普勒超声诊断仪，高频探头频率5.0～13.0 MHz。患者取仰卧位，头部后仰或垫高肩部充分暴露颈前区[1]。平静呼吸，甲状腺二维超声扫查发现结节后，首先用二维超声观察其形态、大小、边界、内部回声、有无钙化等，然后切换到弹性模式，行甲状腺超声弹性成像检查。显示结节并尽量固定探头位置，手持探头在结节部位做微小运动，使显示屏压力指示条的数字控制在3～4[2]，并使感兴趣区域大于结节的2～3倍[3]，用双幅实时显示功能动态观察声像图，对甲状腺结节进行弹性分级。

3.4.1 TLR4 expression of L-02 cells. Removed the fixed cover glass; rinsed with PBS for 3 × 3 min; treated with 3% H2O2 (at room temperature) for 15 min; rinsed with PBS for 3 × 3 min; serum was blocked at 37℃ for 20 min; took out and dried the blocking solution (not rinsed), rabbit anti-TLR4 monoclonal antibody (1∶100) (PBS diluted) at 37℃ for 2 h; rinsed with PBS for 3 × 3 min; secondary antibody incubation at 37℃ for 1 h; rinsed with PBS for 3 × 3 min; SABC incubation at 37℃ for 20 min; rinsed with PBS for 3 × 5 min; DAB developed, stopped staining, and sealed the cover glass. Under the light microscope, the positive cell area and the integral optical density were of each section were analyzed using NIS-Elements Basic Research image acquisition and analysis system.

3.1 Culture of L-02 cells The freezing tube of L-02 hepatocyte cell lines was taken out from -80℃ cryogenic refrigerator, immediately placed in a 37℃ water bath, shaken constantly, frozen stock solution was rapidly melted, immediately added to 1640 culture medium containing 10% fetal bovine serum (preheated at 37℃), centrifuged (at 1 000 rpm for 5 min), discarded the supernatant, added the mixed PBS solution and blown evenly, centrifuged (at 1 000 rpm for 5 min), discarded the supernatant again, added the complete culture medium and blown evenly and centrifuged again, discarded the supernatant, added the complete culture medium to suspend the cells again, added to the culture flask, placed in CO2 incubator, changed the solution every other day, subcultured every two days, and subcultured for 4-5 generations to the logarithmic growth phase for experiment.

3.4 Effects of Saikosaponon d and Baicalin on expression of TLR3 and NFκBp65 of CCl4 injured L-02 cells The L-02 hepatocytes in logarithmic growth phase were counted through trypsin digestion, incubated with cell concentration of 2×105 pcs/mL on the 6-well plate covered with cover glass in advance, cultured for 24 h and then removed the supernatant, (after CCl4 intervention, removed the supernatant), added drugs and cultured for 48 h and removed the supernatant, fixed with 4% formaldehyde overnight, and took out the cover glass for immunohistochemistry experiment.

3.4.2 NFκBp65 expression of L-02 cells. The rabbit anti-NFκBp65 monoclonal antibody (1∶1 00) was used and the specific method was the same as the above.

3.5 Statistical method The experimental data were expressed with the standard deviation of the mean value and SARS software was used for the analysis. First, the homogeneity of variance was tested, then one way-ANOVA was carried out. In case of deviation, Student-Newman-keuls (SNK) was tested, and P<0.05 indicated statistically significant.

4 Results

4.1 Effects of Saikosaponon d and Baicalin on proliferation of L-02 cells Compared with the blank control group, the proliferation of cells in CCl4 intervention group was significantly inhibited. Saikosaponon d and Baicalin could promote the proliferation of CCl4 injured cells, and 1.75 μg/mL Saikosaponon d and Baicalin group showed the most significant proliferation effect, as listed in Table 1.

Table 1 Effects of Saikosaponon d and Baicalin on the growth of CCl4 injured cells±s, n=8)

GroupDoseODvalue Blankcontrolgroup-0．69±0．03∗∗CCl4interventiongroup8．00mmol/L0．37±0．01SaikosaponondandBaicalingroup2．25μg/mL0．42±0．04SaikosaponondandBaicalingroup2．00μg/mL0．52±0．02SaikosaponondandBaicalingroup1．75μg/mL0．59±0．05∗∗SaikosaponondandBaicalingroup1．5μg/mL0．55±0．02∗SaikosaponondandBaicalingroup1．25μg/mL0．50±0．04SaikosaponondandBaicalingroup1．00μg/mL0．41±0．02

Note: compared with CCl4 intervention group **P<0.01 *P<0.05.

4.2 Effects of Saikosaponon d and Baicalin on AST and ALT levels of CCl4 injured L-02 cell culture medium According to the experimental results, the AST and ALT levels of CCl4 intervention group were higher than that of the blank control group, both 1.75 μg/mL and 1.50 μg/mL of Saikosaponon d and Baicalin had effects of reducing the ALT and AST levels, and the 1.75 μg/mL has more significant effects, as shown in Table 2.

Table 2 Effects of Saikosaponon d and Baicalin on AST and ALT levels of CCl4 injured±s, n=8)

GroupDoseALT∥U/LAST∥U/LBlankcontrolgroup- 0．170±0．006∗∗0．204±0．007∗∗CCl4interventiongroup8．00mmol/L 0．264±0．0030．297±0．012SaikosaponondandBaicalingroup1．75μg/mL 0．185±0．005∗∗0．221±0．007∗∗SaikosaponondandBaicalingroup1．50μg/mL 0．197±0．017∗0．235±0．006∗SaikosaponondandBaicalingroup1．25μg/mL 0．215±0．0170．285±0．006

Note: compared with CCl4 intervention group **P<0.01 *P<0.05.

4.3 Effects of Saikosaponon d and Baicalin on expression of TLR3 and NFκBp65 of CCl4 injured L-02 cells The experimental results showed that the positive expression of both TLR4 and NFκBp65 in L-02 cells was in the cytoplasm, which was stained brown and the nuclei were not stained. After CCl4 intervention, the proliferation of L-02 cells decreased and most of the hepatocytes showed a nearly round shape, and the average positive expression of TLR4 and NFκBp65 of single cell increased. Both 1.75 μg/mL and 1.50 μg/mL of Saikosaponon d and Baicalin had effects of reducing the expression of TLR4 and NFκBp65, as shown in Table 3 and Fig.1 and Fig.2.

Table 3 Comparison of expression area of TLR4 and NFκBp65 in cells of each±s, n=8)

GroupDoseTLR4∥%NFκBp65∥%Blankcontrolgroup-19．82±5．24∗∗18．64±5．64∗∗CCl4interventiongroup8．00mmol/L28．06±5．4332．81±0．57SaikosaponondandBaicalingroup1．75μg/mL20．25±4．68∗∗19．73±4．25∗∗SaikosaponondandBaicalingroup1．50μg/mL21．97±8．94∗23．89±2．51∗SaikosaponondandBaicalingroup1．25μg/mL25．88±6．5728．02±3．77

Note: compared with CCl4 intervention group **P<0.01 *P<0.05.

Note: 1. Control group; 2. CCl4 injured cells; 3. Saikosaponon d and Baicalin 1.75 μg/mL; 4. Saikosaponon d and Baicalin 1.5 μg/mL; 5. Saikosaponon d and Baicalin 1.25 μg/mL

Fig.1 Effects of Saikosaponon d and Baicalin on NFκBp65 expression of L-02 cells (×400)

Note: 1. Control group; 2. CCl4 injured cells; 3. Saikosaponon d and Baicalin 1.75 μg/mL; 4. Saikosaponon d and Baicalin 1.5 μg/mL; 5. Saikosaponon d and Baicalin 1.25 μg/mL

Fig.2 Effects of Saikosaponon d and Baicalin on TLR4 expression of L-02 cells (×400)

5 Discussions

Related studies[2-3] have shown that in a variety of liver injury, the invivo TLR4-NFκB signaling pathway-mediated inflammatory response increased, thus there is a close relationship between TLR4-NFκB signaling pathway abnormalities and the liver injury[4-7]. The previous study of our team has shown that the compatibility of Saikosapon d and Baicalin can reduce the liver fibrosis injury of rats, the mechanism of which may be connected with the regulation of TLR4-NFκB signaling pathway. The effects of Saikosapon d and Baicalin on prevention of liver injury are clear[8], but its effective component and action mechanism is still not clear. Saikosapon d and Baicalin are essential active components of B. chinense DC. and Scutellariabaicalensis Georgi. Studies have shown that both have good effects of anti-liver fibrosis[9-12]. On the basis of the previous studies, using the compatibility of Saikosapon d and Baicalin, intervened CCl4 injured hepatocyte lines L-02, we discussed effective components and action mechanism of B. chinense DC. and S. baicalensis Georgi. in preventing liver injury.

The experiment showed that CCl4 obviously leads to acceleration of apoptosis of L-02 cells, reduces the cell activity; ALT and AST significantly increased in cell culture medium, certain liver injury occurred; through intervention of 1.75, 1.5, 1.25 μg/mL of Saikosapon d and Baicalin, 1.75 μg/mL and 1.5 μg/mL could significantly enhance the proliferation of injured L-02 cells and ALT and AST significantly reduced in cell culture medium, and 1.75 μg/mL has more significant effect. The results of immunohistochemistry test showed that the expression of TLR4 and NFκB in L-02 cells were significantly increased after CCl4 intervention, and the expression of TLR4 and NFκB was significantly decreased after the intervention of Saikosapon d and Baicalin, and 1.75 μg/mL has more significant effect. In conclusion, the compatibility of Saikosapon d and Baicalin has a certain effect of reducing liver injury, its mechanism may be related to the regulation of TLR4-NFκB pathway, and deeper mechanisms need further study.

References

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