Research Status of Serum Pharmacology and Serum Pharmacochemistry of Traditional Chinese Medicine

Serum pharmacochemistry of traditional Chinese medicine has become a more accurate and rapidly developing reasonable way to analyze the effective material basis of traditional Chinese medicine in recent years. Through this method, we can select very complex Chinese medicine components and clarify the effective substances consistent with the main functions, and then separate and purify effective ingredients in traditional Chinese medicine, so as to explain the material basis of the drug effect and prove the rationality and principle of action of Chinese medicine compounds. It can show the multi-component and multi-target characteristic of traditional Chinese medicine, and after analyzing the migrating components of serum, we can explore the direct effective substances that act on the patient’s body, so as to accelerate and accurately complete the in-depth study of the effective substances of Chinese medicine.

Application of Serum Pharmacology in Evaluating the Antitumor Effect of Fuzheng Yiliu Decoction(扶正抑瘤方) from Chinese Medicine

Objective： To investigate the feasibility of serum pharmacology in evaluating the antitumor effect of Chinese medicine （CM） of Fuzheng Guben （扶正固本, supporting the healthy energy and strengthening the body＇s resistance to pathogens）, the effects of Fuzheng Yiliu Decoction （扶正抑瘤方, FYD）, a typical prescription of Fuzheng Guben, on proliferation and apoptosis of hepatoma cells in vitro were observed by two methods with serum pharmacology and traditional pharmacology, respectively. Methods： HepG2 cells were treated with FYD- containing serum or crude FYD extract in vitro. The proliferation rate was determined by methyl thiazolyl tetrazolium （MTT） assay. Cell cycle and apoptosis rate was performed by flow cytometry. And the levels of interleukin-2 （IL-2） and tumor necrosis factor α （TNF-α） in FYD-containing serum were detected by radioimmunoassay. Results： FYD-containing serum remarkably inhibited proliferation and induced apoptosis of hepatoma cells at least by promoting the production of IL-2 and TNF- α in vivo. On the contrary, crude FYD extract promoted the proliferation and did not induce cell apoptosis. Conclusion： The results by serum pharmacology were accordant with those of our previous animal and clinical trials which indicates that serum pharmacology is a reasonable and feasible method for the evaluation of the antitumor effect of herbs of Fuzheng Guben.

Serum containing Tongqiaohuoxue decoction suppresses glutamate-induced PC12 cell injury

Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctonus, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate （12.5 mM） was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na＋-K＋-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase I1. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.

Effect of Jinmaitong (筋脉通) Serum on the Proliferation of Rat Schwann Cells Cultured in High Glucose Medium

Objective： To investigate the effect of Jinmaitong （筋脉通,JMT） serum on the proliferation of rat Schwann cells （SCs） primarily cultured in high glucose medium. Method： SOs were primarily cultured in Dulbecco＇s minmum essential medium （DMEM control）, 50 mmol/L glucose medium （50 mmol/L Glu）, 75 mmol/L glucose medium （75 mmol/L Glu）, as well as 50 mmol/L glucose medium, with different concentrations of JMT serum （undiluted, 1：2 diluted and 1：8 diluted） and Neurotropin （Ntp）, respectively. The proliferation of SCs under different conditions was detected by MTT. Result： SCs grew exuberantly in DMEM within 24-72 h, but slowed down at 96 h. The proliferation of SCs was inhibited in 50 mmol/L Glu and 75 mmol/L Glu after cultures of 48, 72 and 96 h, which showed that both were significantly different compared to the control group （P〈0.01）. The inhibition was more significant in 75 mmol/L Glu than in 50 mmol/L Glu （P〈0.05）. Spearman＇s rho analysis revealed that the proliferation of SCs had a negative correlation with the concentration of glucose （r=-0.471, P〈0.01）. Excluding the time factor, partial correlation showed similar results （r =-0.679, P〈0.01）. After 48 h, the proliferation of SCs increased significantly in JMT 1：2 and Ntp compared with 50 mmol/L Glu （control 0.437±0.019, 50 mmol/ L Glu 0.367±0.035, JMT1：2 0.426±0.024, Ntp 0.422±0.013; P〈0.01）, and there were no statistically significant differences among the JMT groups, the Ntp group and the control group （P〉0.05）. Conclusions： The proliferation of SCs was inhibited in high glucose medium, and the inhibition was reduced by different concentrations of JMT serum, especially at JMT1：2.

Effects of Chinese Herbal Medicine Serum on the Apoptosis of Sinoatrial Node Cells Induced by Simulated Ischemia-reperfusion

Objective:To study the effect of Chinese herbal medicine Kangxin Fumai Granule(康心复脉颗粒 Granule for heart diseases) serum on the primary cultured sinoatrial node(SAN) cell apoptosis induced by simulated ischemia-reperfusion(IR).Methods:The SAN cells removed from SAN tissue of neonatal Wistar rats were cultured and purified with differential attachment and 5'-bromodeoxyuridine(BrdU) treatment.Simulated IR model was adopted.The obtained cells were morphologically observed with inverted microscopy.By using the method of serum pharmacology,the cell apoptosis was measured with TUNEL staining qualitatively and with flow cytometry quantitatively.Results:Three kinds of cells were observed in the cultured SAN cells:spindle,triangle and irregular.The spindle cells comprised the greatest proportion.The SAN cells in the model group showed moderate positive brown staining in the nucleus,and the apoptosis rate increased significantly compared to that in the control group(P<0.01).While the SAN cells in the Kangxin Fumai Granule high-dose group did not demonstrated positive staining in the nucleus,and the apoptosis rate decreased significantly compared to that in the model group(P<0.05).Conclusion:Of the cells cultured from SAN,the spindle cells were pacemaker cells of SAN in rats.Blockade and/or inhibition of the SAN cell apoptosis might be one of the important mechanisms of Kangxin Fumai Granule in preventing and treating sinoatrial injury induced by simulated IR.

Effects of Bushen Tiaochong Recipe (补肾调冲方) Containing Serum on Ovarian Granulosa Cell Proliferation,Steroidogenesis and Associated Gene Expression in Rats

Objective： To observe the effect of Bushen Tiaochong Recipe （补肾调冲方, BSTCR） on rats＇ ovarian granulosa cell （GC） proliferation, steroidogenesis and follicle-stimulating hormone receptor （FSHR）, and insulin-like growth factor-1 （IGF-1） mRNA expression using serum pharmacological method. Methods： Rats＇ GCs were incubated with 10% blank serum （as negative control group）, follicle- stimulating hormone （FSH）-containing serum （S-FSH, as positive control group）, or BSTCR （in different dosages） containing serum （S-BSTCR, as the BSTCR groups） for 48 h. ^3H-TdR incorporation was then performed; DNA was measured to analyze the distribution of GCs in the cell cycle and their proliferation index （PI） using a flow cytometer; estradiol （E2） and progesterone （P） content in the culture fluid were examined by radioimmunoassay; and levels of FSHR and IGF-1 mRNA expression in GCs were measured by real-time RT-PCR. Results： A dose-dependent increase of ^3H-TdR incorporation in GC was shown in the BSTCR groups. Cells in Go/G1 phase had markedly less, while those in S phase had a significantly higher increase in the BSTCR groups compared with the negative control group. A high value of PI was also shown in the BSTCR groups, especially in the high dose group where the influence of cell proliferation was stronger than that in the positive control group. The levels of E2 and P in the BSTCR groups of all dosages were significantly higher than those in the negative control group, and did not show any significant difference compared with those in the positive control group. Levels of FSHR and IGF-1 mRNA expression in the BSTCR groups increased in a dose-dependent manner at levels higher than those in the negative control group. Conclusion： S-BSTCR can obviously stimulate the proliferation and steroidogenesis of ovarian GCs.It is speculated that BSTCR could play a regulatory action on ovarian function through two different pathways of endocrine and autocrine by promoting FSHR and IGF-1 mRNA expression.

Zhuanggu Jianxi Decoction(壮骨健膝方) Limits Interleukin-1β-Induced Degeneration Chondrocytes via the Caveolin-p38 MAPK Signal Pathway

Objective： To evaluate the effect of Zhuanggu Jianxi Decoction （壮骨健膝方, ZGJXD） on interleukin- 1 β- （IL-1 β- ）-induced degeneration of chondrocytes （CDs） as well as the activation of caveolin-p38 mitogen- activated protein kinase （MAPK） signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis. Methods： Serum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum （ZGJXD-S） was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL- l β-stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type H collagen immunocytochemistry. After IL-1 β-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 （a specific inhibitor of p38 MAPK） for 48 h, caveoUn-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL- 1β-, tumor necrosis factor ~ （TNF-oL）, matrix metalloproteinase 3 （MMP-3） and MMP-β were examined by real-time polymerase chain reaction. Results： IL-1 β- stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 β, TNF-β, MMP-3 and MMP-β. However, the IL-1 β-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs. Conclusion： ZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.

Xiongshao Capsule(芎芍胶囊) Promotes Angiogenesis of HUVEC via Enhancing Cell Proliferation and Up-regulating the Expression of bFGF and VEGF

Objective： To evaluate the angiogenic effect of the Xiongshao capsule （芎芍胶囊, XSC） in human umbilical vein endothelial cells （HUVEC）, and to investigate the possible molecular mechanisms mediating its biological effect. Methods： Serum pharmacology was applied in this study, in which different doses of XSC were administrated to rats orally and then XSC-containing serum （XSC-S） was collected for the following in vitro experiments. The viability of HUVEC was determined by the 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay. Cell density was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with propidium iodide staining was performed to determine cell cycle phase. Cell migration was determined by wound-healing method. Capillary tube formation by HUVEC was examined using ECMatrix gel- based assay. Vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） expression levels were measured by reverse transcription polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbant assay （ELISA） analyses. Results： XSC-S dose-dependently stimulated proliferation of HUVEC by promoting the cell cycle G1 to S progression. In addition, XSC-S treatment dramatically increased the migration and capillary tube formation of HUVEC in a dose-dependent manner. Moreover, XSC-S enhanced the expression of VEGF and bFGF at both mRNA and protein levels. Conclusion： XSC can promote several features of angiogenesis in endothelial cells through up-regulating the expression of bFGF and VEGF, suggesting that XSC may be a potential novel therapeutic agent for the treatment of ischemic heart diseases.