Anti-depression targets and mechanism study of Kaixin San / 中国中药杂志

This study identified the anti-depression targets of Kaixin San(KXS) in the brain tissue with "target fishing" strategy, and explored the target-associated pharmacological signaling pathways to reveal the anti-depression molecular mechanism of KXS. The Balb/c mouse model of depression was established by chronic unpredictable mild stress(CUMS) and the anti-depression effect of KXS was evaluated by forced swimming test and sucrose preference test. KXS active components were bonded to the benzophenone-modified magnetic nanoparticles by photocrosslinking reaction for capturing target proteins from cortex, thalamus and hippocampus of depressive mice. The target proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS). The enrichment analysis on signaling pathways was performed by Cytoscape. The potential biological functions of targets were verified by immunohistochemistry and Western blot assay. The results showed that KXS significantly improved the behavioral indexes. There were 64, 91, and 44 potential targets of KXS identified in cortex, thalamus, and hippocampus, respectively, according to the target identification experiment. The functions of these targets were mainly associated with vasopressin-regulated water reabsorption, salmonella infection, thyroid hormone synthesis, and other signaling pathways. Besides, the results of immunohistochemistry and Western blot showed that KXS up-regulated the expressions of argipressine(AVP) in the cortex, heat shock protein 60(HSP60), cytochrome C oxidase 4(COX4), and thyrotropin-releasing hormone(TRH) in the thalamus, and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and nuclear factor kappa B(NF-κB) p65 in the thalamus. Therefore, KXS may exert anti-depression effect through regulating vasopressin signaling pathway in the cortex and inflammation, energy metabolism, and thyroid hormone signaling pathways in the thalamus, and the effect of KXS on hippocampus is not significant.

Extract of Fructus Schisandrae chinensis Inhibits Neuroinflammation Mediator Production from Microglia via NF-κ B and MAPK Pathways / 中国结合医学杂志

OBJECTIVE@#To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis (EFSC) on lipopolysaccharide (LPS)-induced BV-2 cells and the possible involved mechanisms.@*METHODS@#Primary cortical neurons were isolated from embryonic (E17-18) cortices of Institute of Cancer Research (ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group (treated with 1 μg/mL LPS only) and EFSC groups (treated with 1 μg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide (MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide (NO) and interleukin-6 (IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence.@*RESULTS@#EFSC (200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression in LPS-induced BV-2 cells (P<0.01 or P<0.05). EFSC (200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia (P<0.01). In addition, EFSC alleviated cell apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium (P<0.01). The mechanistic studies indicated EFSC could suppress nuclear factor (NF)-?B phosphorylation and its nuclear translocation (P<0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase (MAPK) pathway (P<0.01 or P<0.05).@*CONCLUSION@#EFSC acted as an anti-inflammatory agent in LPS-induced glia cells. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.

Neuroprotective effects and mechanism of ethanol extract of Cistanche tubulosa against oxygen-glucose deprivation/reperfusion / 中国中药杂志

To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. RESULTS:: showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.

Method for active ingredients' in vivo target identification of traditional Chinese medicine using magnetic nanoparticles / 中国中药杂志

Target identification is an important prerequisite for the study of medicine action mechanism. Currently,drug target identification is mostly based on various cell models in vitro. However,the growth microenvironment,nutrition metabolism,biological properties as well as functions are quite different between in vitro cell culture and physiological environment in vivo; wherefore,it is a challenging scientific issue to establish an effective method for identifying drug targets in vivo condition. In this study,we successfully prepared a kind of magnetic nanoparticles( MNPs) which can be chemically modified by the hydroxyl structure of natural bioactive compound echinacoside( ECH) via the epoxy group label on the surface of MNPs. Therefore,organ-selective and recoverable nanoscale target-recognizing particles were prepared. We then intravenously injected the ECH-binding MNPs into rats and distributed them to specific organs in vivo. After cell endocytosis,ECH-binding MNPs captured target proteins in situ for further analysis. Based on this method,we discovered several potential target proteins in the spleen lysates for ECH,and preliminarily clarified the immuno-regulation mechanism of ECH. Collectively,our strategy developed a proof-of-concept technology using nanoparticles for in vivo target identification,and also provided a feasible approach for drug target prediction and pharmacological mechanism exploration.

TCM chemical biology--emerging interdiscipline of "TCM chemistry" and "biology" / 中国中药杂志

Traditional Chinese medicine(TCM) is a research area with highly original innovation features,and is also a Chinese name card to the world. However,TCM owns a unique theoretical system which is quite different from western modern medicine,leading to an awkward situation of deficient modern social identity as well as poor international spread. Therefore,how to establish a research strategy in line with the characteristics of TCM itself to systematically interpret the unique scientific connotation of TCM is always a public hot topic. Based on persistent practical exploration and scientific consideration in TCM,our group firstly promoted the concept of traditional Chinese medicine chemical biology(TCM chemical biology,TCMCB). The major idea of TCMCB is to clarify the nature of TCM regulating life progress to link TCM to modern medicine by using TCM components as chemical tools. Notably,TCMCB mainly focuses on TCM target identification and TCM-guided disease molecular mechanism exploration,further to clarify the basic law of TCM mediating disease process. Finally,TCMCB-guided scientific studies can help explain TCM theory and promote the developmentof modern innovative drugs based on identified targets using TCM active components. Moreover,TCMCB is of vital importance for investigating the scientific nature of biological progress and the pattern of disease occurrence and development,indicating a key significance for modern life science and medicine. This review introduces the definition of TCMCB as well as its academic thought,research method,technology system and scientific significance,for providing new research ideas and scientific thoughts for TCM development.

Traditional Chinese medicine target identification based on"Target Fishing"strategy / 中国药理学与毒理学杂志

OBJECTIVE Our group mainly focuses on the target identification and pharmacological mechanism study of TCM.We deeply identified the direct targets of the active ingredients in TCM using molecule probe-'Target Fishing' technology in chemical biology, and explored the related signaling pathways to explain the traditional efficiency of TCM. METHODS We synthesized biotin-tagged mole-cule probe by connecting biotin tag to TCM active molecule using PGE as a linker. Then, the biotin-tagged molecule probe was bound to the surface of solid beads by strong biotin-avidin interaction. Thus, the molecule probe-bound beads were mixed with cell lysates to capture the potential targets and identified by MS.RESULTS Our study found that SA which was an anti-inflammatory compound-could selectively bind to IMPDH2 in microglial cells,and SA showed weaker anti-inflammatory effect on IMPDH2-knock down microglial cells,suggesting IMPDH2 as a key anti-inflammatory target for SA.Ad-ditionally,handelin was a key anti-inflammatory compound.We identified the target protein of handelin as Hsp70 from microglial cells using target pull-down technology. Moreover, handelin showed weaker anti-inflammatory effect on Hsp70-knock down microglial cells,revealing that Hsp70 was the direct anti-inflammatory target of handelin. CONCLUSION Our study provided methodology references for TCM target identification in the future, and also showed a new insight for exploring the pharmacological mechanism of TCM active ingredients.More importantly,we can perform scientific annotation for TCM efficiency by clarifying the biological functions of each target protein,showing important significance on modernization and internationalization of TCM.

Pharmacological targets identification and efficacy analysis of phenylethanoid glycosides from Cistanches Herba based on “target fishing” strategy / 中草药

Objective Cistanches Herba is a kind of tonic traditional Chinese medicine with several therapy effects including tonifying kidney-yin, anti-dementia, anti-aging and relaxing bowel. Phenylethanoid glycosides (PhGs) are the major effective components in C. tubulosa. However, there were no further studies on molecular pharmacologic mechanisms due to its complex components and mechanism diversity of action in PhGs till to now. The aim of this study was to investigate the target protein groups and related mechanisms associated with PhGs in anticerebral ischemia-reperfusion injury. Methods The middle cerebral artery occlusion (MCAO) model was established in rats, and the protective effects of PhGs on cerebral ischemia-induced injuries were determined. A kind of solid bead whose surface was cross-linked with PhGs was prepared to capture the target proteins from brain tissue lysates. The target proteins were further identified with LC-MS/MS. Results PhGs significantly inhibited cerebral ischemia-induced injuries by reducing ischemia size and rat behavioral scores and elevated the SOD levels in rat brain tissues. Eighteen target proteins were identified based on “target fishing” strategy and divided into 9 kinds according to their biological functions, including anti-oxidation, ion channel, immunoregulation, cell survival and cytoskeleton, etc. Conclusion These findings reveal the potential pharmacological mechanisms of PhGs in anti-dementia, fatigability alleviating, anti-tumor, immunoloregulation, etc, and also present a promising technology for investigating the complicated pharmacological mechanisms of traditional Chinese medicine.

Anti-tumor target identification and molecular mechanism study of total saponins from Albizia julibrissin / 中国中药杂志

Dried stem bark from Albizia julibrissin(AJ) is a common traditional Chinese herb with several therapy effects including insomnia, anxiety and anti-tumor. Recently, the anti-tumor effect and mechanism studies of AJ have drawn much attention; however, there are still some troubles in chemical composition separation, which leads to the difficulties in pharmacological research of AJ. In this study, we firstly confirmed the proliferation inhibitory effect of total saponins from AJ(TSAJ)on human hepatocarcinoma(HepG2) cells, and also tested the apoptosis induction effect of TSAJ. Then, we successfully captured the potential target proteins from HepG2 lysates by using TSAJ-modified solid beads, and identified the target proteins by LC-MS/MS. Finally, we confirmed 5 target proteins including Exportin-2, Beta-actin-like protein 2, Myosin-9, Protein transport protein Sec61 subunit beta,and Cytochrome c oxidase copper chaperone, which are responsible forcell apoptosis, proliferation, differentiation andmigration. In summary, our findings elucidate the potential anti-tumor mechanism of TSAJ from the direct target proteins, and provide a new insight for exploring the pharmacological mechanism of traditional Chinese medicine.

Identification and function analysis of target group for cardioprotection of Baoyuan decoction / 中国中药杂志

Baoyuan decoction (BYD) is a well-known traditional Chinese medicine formula for coronary heart disease with Qi deficiency. However, the detailed pharmacological mechanism of BYD is still unknown because of its complicated chemical compositions. In this study, we synthesized a kind of solid beads with benzophenone groups on its surface. Benzophenone can be activated and chemically cross-linked with the C-H bonds of the chemical compositions in BYD (BYD beads) under UV activation. We thus captured all the target proteins from mouse heart tissue lysates by using BYD beads. Based on proteomics analysis, we discovered totally 46 potential binding target proteins, most of which were located in mitochondria. KEGG analysis revealed that these target proteins were mainly associated with TCA cycle and amino acid metabolism signaling pathways, suggesting that the cardioprotection of BYD might be associated with regulating mitochondrial function and energy production. Moreover, JC-1 staining analysis also confirmed the protective effect of BYD on mitochondrial damage. In summary, our findings elucidated the potential mechanism of BYD on cardioprotection through "target fishing" strategy, and further explained its traditional efficacy in the molecular level. In addition, we also provide an approach for investigating the target group of complicated compositions in Chinese herbal formula. This novel method may provide a methodological reference for exploring the pharmacological mechanism of traditional Chinese formula in the future.

Current status and challenging of direct target study of traditional Chinese medicine complex system based on solid coupling beads / 中国中药杂志

Traditional Chinese medicine(TCM) is a complex system with multiple chemical compositions. The most significant character of TCM is that the chemical compositions interact with each other by multi-target synergism to treat diseases. Previous reports mainly focused on the investigation of single signaling pathway detection or the phenotypic analysis of proteomics difference; however, no studies have been conducted on the identification of direct targets of TCM. Therefore, it is difficult to analyze the molecular mechanism of traditional Chinese medicine from the target source, and it is difficult to explain its traditional efficacy scientifically, thus seriously affecting its clinical application and internationalization. In this article, we discussed the methodology for the identification of direct TCM targets(groups), and presented the strategy for preparation of TCM chemical composition solid coupling beads, as well as of enrichment and identification strategy of target proteins based on photosensitive coupling technique. We also discussed the advantages and limitations of this strategy, and put forward some new ideas for the future developments. We hope this article can provide some guidance and reference significance for the researchers on TCM pharmacology study, especially on target identification.

Progress of methodology for identifying target protein of natural active small molecules / 中国中药杂志

Drug targets are special molecules that can interact with drugs and exert pharmacological functions in human body. The natural active small molecules are the bioactive basis of traditional Chinese medicine, and the mechanism study is a hot topic now, especially for the identification of their target proteins. However, little progress has been made in this field until now. Here, we summarized the recent technologies and methods for the identification of target proteins of natural bioactive small molecules, and introduced the main research methods, principles and successful cases in this field. We also explored the applicability and discussed the advantages and disadvantages among different methods. We hope this review can be used as a reference for the researchers who engaged in natural pharmaceutical chemistry, pharmacology and chemical biology.

Triterpenoid saponins from Ilex mamillata C.Y. Wu ex C.J. Tseng.

Two new triterpenoid saponins, ilemaminosides A and B (1 and 2) along with six known saponins (3-8) were isolated from 70% ethanolic extract of the leaves of Ilex mamillata C.Y. Wu ex C.J. Tseng. The new saponins were characterised as 3-O-α-L-arabinopyranosyl-ilexgenin B (1) and 3-O-ß-D-glucopyranosyl-(1 → 3)-α-L-arabinopyranosyl-ilexgenin B (2). The structures of compounds 1 and 2 were elucidated on the basis of the chemical and spectroscopic methods, and the structures of known compounds were identified by comparison of their spectroscopic data with those reported in the literature. The compounds showed inhibitory activities in anti-inflammatory assay in vitro with IC(50) values in the range 25.37-38.33 µg mL(-1).

Protective effect of cerebrospinal fluid containing Jiawei Wuzi Yanzong formula on beta-amyloid protein-induced injury of hippocampal neurons / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the protective effects of cerebrospinal fluid (CSF)-containing Jiawei Wuzi Yanzong formula (CSF-C) on beta-amyloid protein-induced injury of hippocampal neurons.</p><p><b>METHODS</b>Primary hippocampal neurons were isolated and cultured for 7 days in vitro. CSF-C was prepared by cerebrospinal fluid extracted from SD rats fed continuously with various components of Jiawei Wuzi Yanzong formula (total formula, total flavonoids or total polysaccharides) respectively. The viability, morphological change, apoptotic rate and apoptosis-related proteins (caspase-3, PARP, Bcl-2, Bcl-XL, JNK1/2 and p38 MAPK) of neurons were detected in different groups: the untreated normal group, the Abeta(25-35)-treated group, and the CSF-C protected groups (co-acted by CSF-C and 20 micromol/L of Abeta(25-35)), respectively.</p><p><b>RESULTS</b>CSF-C showed significant neuro-protective effect, and the protection of CSF-C contained total flavonoids or total polysaccharides was significantly greater than that contained total formula (P < 0.05 or P < 0.01). Moreover, the effects of CSF-C contained various components on apoptosis-related proteins were different.</p><p><b>CONCLUSION</b>Some flavonoid and polysaccharide components in Jiawei Wuzi Yanzong formula can pass through the blood brain barrier and protect neurons from beta-amyloid protein-induced neuron injury to some extents.</p>