[Effects of tetramethylpyrazine on pharmacokinetics in plasma and brain dialysate and neuropathic pain in rat model of spared sciatic nerve injury].

This study aims to explore the effects of tetramethylpyrazine(TMP) on pharmacokinetics in plasma and brain dialysate and neuropathic pain in the rat model of partial sciatic nerve injury(SNI), and to investigate the correlation between the analgesic effect of TMP and its concentrations in the plasma and brain dialysate. Male SD rats were randomized into Sham, SNI, and SNI+TMP groups. Mechanical stimulation with von frey filaments and cold spray method were employed to evaluate the mechanical sensitivity and cold sensitivity of rats. Another two groups, Sham+TMP and SNI+TMP, were used to intubate the common jugular vein and implant microdialysis probes into the anterior cingulate gyrus(ACC), respectively.After intraperitoneal injection of TMP at a dose of 80 mg·kg~(-1), automatic blood collection and intracerebral microdialysis(perfusion rate of 1 µL·min~(-1)) systems were used to collect the blood and brain dialysate for 24 h. HSS T3 C_(18) reversed-phase chromatographic column(2.1 mm×50 mm, 2.5 µm) was used for liquid chromatographic separation. Gradient elution was carried out with the mobile phase of methanol-water(containing 0.005% formic acid) at a flow rate of 0.25 mL·min~(-1). Electrospray ion source was used for mass spectrometry, and the scanning mode was multi-reaction monitoring under the positive ion mode. The ion pairs for quantitative analysis were TMP m/z 137/122 and aspirin m/z 179/137, respectively. DAS 2.11 was used to calculate the pharmacokinetic parameters. The optimal time of TMP to exert the analgesia effect and inhibit cold pain sensitivity was 60 min after treatment. The TMP in the plasma and brain dialysate of SNI rats showed the T_(max) of 15 min and 30 min, the C_(max) of(2 866.43±135.39) and(1 462.14±197.38) µg·L~(-1), the AUC_(0-t) of(241 463.30±28 070.31) and(213 115.62±32 570.07) µg·min·L~(-1), the MRT_(0-t) of(353.13±47.73) and(172.16±12.72) min, and the CL_Z of 0.73 and 0.36 L·min·kg~(-1), respectively. The analgesic effect of TMP had a significant correlation with the blood drug concentration in the ACC, which indicated that this method was suitable for the detection of TMP in rat plasma and brain dialysate. The method is accurate, reliable, and sensitive and can realize the important value of the application of correlation analysis theory of "automatic blood collection-microdialysis/PK-PD" in the research on neuropathic pain.

Shenlian extract protected ox-LDL-loaded macrophages against ER stress by promoting LAL-LXRα mediated cholesterol flux.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlian (SL) extract is consisted of extracts from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm.f.) Nees, two herbs commonly used in Chinese clinical formula to treat atherosclerosis by removing blood stasis and clearing away heat. Pharmacologically, the anti-atherosclerotic effects of these two herbs are related to unresolved inflammation and the macrophage anergy or apoptosis in lesions led by the lipid flux blockage and ER stress. However, the deeper understanding of SL extract in protecting macrophage in plaques remains unknown. AIM OF THE STUDY: This study aimed to investigate the underlying mechanism of SL extract in protecting ER-stressed macrophages from apoptosis in atherosclerosis. METHODS: The ApoE-/- atherosclerotic mice model and ox-LDL loaded macrophages model were established to assess the effect of SL extract on ER stress in vivo and in vitro. Key markers related to ER stress in plaque were determined by immunohistochemical staining. Proteins involved in apoptosis and ER stress in macrophages loaded by ox-LDL were assessed by Western blot. ER morphology was observed by electron microscope. Lipid flux was temporally and quantitatively depicted by Oil red staining. The LAL and LXRα were blocked by lalistat and Gsk 2033 respectively to investigate whether SL extract protected the function of macrophages by the activation of LAL-LXRα axis. RESULTS: Our study reported that, in ApoE-/- atherosclerotic mice, SL extract effectively relieved ER stress of carotid artery plaque. In lipid-overloaded macrophage models, SL extract significantly alleviated ER stress by promoting cholesterol degradation and efflux, which finally prevented apoptosis of foam cells induced by ox-LDL. Blockage of ER stress by 4-Phenylbutyric acid (4-PBA), an inhibitor of Endoplasmic Reticulum (ER) stress, largely attenuated the protective effects of SL extract on macrophage. By utilizing the selective antagonists against both LAL and LXRα, this study further revealed that the beneficial effects of SL extract in macrophages was dependent on the proper functionalization of LAL-LXRα axis. CONCLUSIONS: By highlighting the therapeutic significance of macrophage protection in resolving atherosclerosis inflammation, our study pharmacologically provided convincing mechanistic evidence of SL extract in the activation LAL-LXRα axis and revealed its promising potential in the promotion of cholesterol turnover and prevention of ER stress induced apoptosis in lipid-loaded macrophages.

Pharmacokinetic Differences of Wuji Pill Components in Normal and Chronic Visceral Hypersensitivity Irritable Bowel Syndrome Rats Attributable to Changes in Tight Junction and Transporters.

The Wuji pill, also called Wuji Wan (WJW), is an effective traditional medicine for the clinical treatment of irritable bowel syndrome (IBS). It is principally composed of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, and Radix Paeoniae Alba. There have been no reports on the pharmacokinetics of WJW on IBS. Because it is more meaningful to study pharmacokinetics in relation to specific pathological conditions, our study investigated the pharmacokinetic differences of five representative components (berberine, palmatine, evodiamine, rutaecarpine, and paeoniflorin) in normal rats and chronic visceral hypersensitivity IBS (CVH-IBS) model rats after single dose and multiple doses of WJW using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Transmission electron microscopy, immunohistochemistry, and immunofluorescence were used to explore mechanisms behind the pharmacokinetic differences in terms of tight junction proteins (Occludin and ZO-1), myosin light chain kinase (MLCK), and transporters including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and multidrug resistance associated protein 2 (MRP2) in rat colons. After a single dose, for all components except rutaecarpine, significant differences were observed between normal and model groups. Compared with normal group, T1/2 and AUC0-t of berberine and palmatine in model group increased significantly (562.5 ± 237.2 vs. 1,384.9 ± 712.4 min, 733.8 ± 67.4 vs. 1,532.4 ± 612.7 min; 5,443.0 ± 1,405.8 vs. 9,930.8 ± 2,304.5 min·ng/ml, 2,365.5 ± 410.6 vs. 3,527.0 ± 717.8 min·ng/ml), while Cl/F decreased (840.7 ± 250.8 vs. 397.3 ± 142.7 L/h/kg, 427.7 ± 89.4 vs. 288.9 ± 114.4 L/h/kg). Cmax and AUC0-t of evodiamine in model group increased significantly (1.4 ± 0.6 vs. 2.4 ± 0.7 ng/ml; 573 ± 45.3 vs. 733.9 ± 160.2 min·ng/ml), while T1/2, Tmax, Cl/F, and Vd/F had no significant difference. Tmax and AUC0-t of paeoniflorin in model group increased significantly (21.0 ± 8.2 vs. 80.0 ± 45.8 min; 15,428.9 ± 5,063.6 vs. 33,140.6 ± 5,613.9 min·ng/ml), while Cl/F decreased (110.5 ± 48.1 vs. 43.3 ± 9.5 L/h/kg). However, after multiple doses, all five components showed significant differences between normal and model groups. Moreover, these differences were related to tight junction damage and the differential expression of transporters in the colon, suggesting that dose adjustment might be required during administration of WJW in the clinical treatment of IBS.

Network pharmacology analysis and experimental validation to explore the mechanism of Shenlian extract on myocardial ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlian extract (SL), extracted from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm. f.) Nees, has been proved to be effective in the prevention and treatment of atherosclerosis. Recently, we have partially elucidated the mechanisms involved in the therapeutic effects of SL on myocardial ischemia (MI). However, the underlying mechanisms remain largely unclear. AIM OF THE STUDY: This study aims to explore the potential molecular mechanism of SL on MI on the basis of network pharmacology. MATERIALS AND METHODS: First, the main active ingredients of SL were screened in the Traditional Chinese Medicine Integrated Database, and the MI-associated targets were collected from the DisGeNET database. Then, we used compound-target and target-pathway networks to uncover the therapeutic mechanisms of SL. On the basis of network pharmacology analysis results, we assessed the effects of SL in MI rat model and oxygen glucose deprivation model of H9c2 cells and validated the possible molecular mechanisms of SL on myocardial injury in vivo and in vitro. RESULTS: The network pharmacology results showed that 37 potential targets were recognized, including TNF-α, Bcl-2, STAT3, PI3K and MMP2. These results revealed that the possible targets of SL were involved in the regulation of inflammation and apoptosis signaling pathway. Then, in vivo experiments indicated that SL significantly reduced the myocardial infarction size of MI rats. Serum CK-MB, cTnT, CK, LDH, and AST levels were significantly decreased by SL (P < 0.05 or P < 0.01). In vitro, SL significantly increased H9c2 cell viability. The levels of inflammation factors including TNF-α and MMP2 were significantly decreased by SL (P < 0.05 or P < 0.01). TUNEL and Annexin V/propidium iodide assays indicated that SL could significantly decrease the cell apoptotic rate in vivo and in vitro (P < 0.05 or P < 0.01). The remarkable upregulation of anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax protein level further confirmed this result. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the PI3K-AKT and JAK2-STAT3 pathways were significantly enriched in SL. Compared with the model group, SL treatment significantly activated the PI3K-AKT and JAK2-STAT3 pathways in vivo and in vitro according to Western blot analyses. CONCLUSION: SL could protect the myocardium from MI injury. The underlying mechanism may be related to the reduction of inflammation and apoptosis by activating the PI3K/AKT and JAK2/STAT3 pathways.

[Pharmacodynamic substances and therapeutic potential of Wuji Pills:based on UPLC-Q-TOF-MS/MS and network pharmacology].

As a classic prescription, Wuji Pills is composed of Coptidis Rhizoma, Euodiae Fructus Preparata, and stir-fried Paeo-niae Radix Alba at the ratio of 6∶1∶6. The practical application of it is limited compared with other famous Chinese medicine prescriptions. Only one company produces Wuji Pills in China. In this study, ultra-performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used to analyze and identify 26 identical compounds from Wuji Pills and drug-containing plasma of rats. Based on these components, 46 potential targets were screened out with network pharmacology methods, followed by the component-target network construction, Gene Ontology(GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and disease prediction. It was concluded that Wuji Pills acted on core targets such as PTGS2, PTSG1, NCOA2, HSP9 OAD1, and RXRA through magnoflorine, hydroxyevodiamine, daucosterol, and berberine and exerted pharmacodynamic effects through various pathways such as calcium ion signaling pathway, phosphatidylinositol-3-kinase-protein kinase B(PI3 K-Akt) signaling pathway, and vascular endothelial growth factor(VEGF) signaling pathway. Thus, Wuji Pills has therapeutic potential for Alzheimer's disease, diabetes mellitus, myocardial ischemia, and other diseases in addition to the conventional disease(irritable bowel syndrome, IBS). The above research results can provide a reference for the comprehensive interpretation of the pharmacodynamic basis of Wuji Pills and the expansion of clinical application. At the same time, a lot of components in serum and the in vivo transformed and metabolized components of Wuji Pills have similar structure and relative molecular weight. In theory, these components may show additive effects and the competitive/antagonistic effects on the same target. According to the hypothesis of "additive effect of multiple components for a single target" in traditional Chinese medicine, multiple similar components may exert the additive effects on local targets. This study can partly prove the scientificity of this hypothesis and provide laboratory evidence.

Muyin extract inhibits non-small-cell lung cancer growth by inducing autophagy and apoptosis in vitro and in vivo.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer with a higher mortality rate. Both apoptosis and autophagy are crucial processes in the pathophysiology of NSCLC. Muyin extract (MSE) is a combination of Momordica cochinchinensis (Lour.) Spreng seeds and Epimedium brevicornu Maxim extract, with an optimal ratio of 1:1. Our previous research has firstly shown that MSE exerts a good anti-tumor activity, especially for NSCLC. PURPOSE: This study aims to evaluate the inhibitory effect of MSE on NSCLC and explore the underlying mechanism. METHODS: In vitro, cell proliferation was examined by MTT and colony formation. Apoptosis was detected by annexin V-FITC/PI assay while autophagy was assessed by Acridine orange (AO) and Monodansylcadaverine (MDC) staining. In vivo, Lewis lung cancer cell transplanted mice model was established to measure the effect of MSE on tumor growth. Hematoxylin eosin (H & E) staining was used to observe the pathological changes of the tumor after MSE treatment. The apoptosis in tumor tissue was detected by TUNEL assay. Meanwhile, the cellular proliferation marker Ki67 and autophagy marker LC3Ⅱ were observed by immunohistochemistry staining. The IL-4 and IFN-γ concentrations in blood were tested by Elisa. The apoptosis related factors (Bcl-2, Bax Caspase-3, cleaved Caspase-3, Caspase-9 and p53), autophagy marker proteins (Atg-5, Becline-1, LC3Ⅱ/Ⅰand p62) as well as Akt/mTOR pathway were detected by western blotting. RESULTS: Present study showed that MSE greatly inhibited the proliferation of NSCLC in vitro and in vivo, together with apoptotic rate increasing. P53 and cleaved Caspase-3 levels were up-regulated while Bcl-2/Bax ratio, Caspase-3 and Caspase-9 levels were significantly down-regulated treated with MSE. Meanwhile, MSE activated autophagy, Atg-5, Becline-1 as well as the ratio of LC3Ⅱ/Ⅰ were notably up-regulated while p62 was down-regulated after MSE treatment. Importantly, MSE significantly blocked Akt/mTOR pathway, which is a common upstream signal triggered by autophagy and apoptosis. Furthermore, when co-treated with specific autophagy inhibitor, the inhibitory rate and anti-apoptotic Bcl-2 level were significantly reversed. Impressively, MSE remarkably increased IFN-γ/ IL-4 ratio while VP16 did not in animal model, and the inhibition rate in tumor weight after MSE treatment was higher than xiaojin pill. CONCLUSION: Taken together, it is proved that MSE may be a promising oral TCM candidate for NSCLC therapy with immunity improvement. The underlying mechanisms could be associated with the induction of apoptosis and autophagy through blocking Akt/mTOR pathway, meanwhile, it may promote crosstalk between autophagy and apoptosis.

[Mechanism of Chuanxiong Rhizoma intervention on central sensitization of Panx1-Src-NMDAR-2B signaling pathway in neuropathic pain model rats].

Excitatory toxicity(ET) is an important factor of neuropathic pain(NPP) induced by central sensitization(CS), and the association of pannexin-1(Panx1)-Src-N-methyl-D-aspartate receptor subunit 2 B(NMDAR-2 B) is an important new pathway for ET to initiate CS. The present study confirmed whether the central analgesic effect of Chuanxiong Rhizoma extract(CRE) was achieved through the synchronous regulation of the brain and spinal pathways of Panx1-Src-NMDAR-2 B. In this study, dynamic and simulta-neo-us microdialysis of the brain and spinal cord in vivo combined with behavioristics, high performance liquid chromatography(HPLC)-fluorescence detection, microdialysis analysis(ISCUS~(flex)), ultrasensitive multifactorial electrochemiluminescence immunoassay, ELISA, and Western blot was employed to investigate the protein expression of NMDAR-2 B, Src, and Panx1, extracellular excitatory amino acids, cytokines, energy metabolites, and substance P in spinal dorsal horn(SDH) and anterior cingulate cortex(ACC) after CRE intervention with the rat model of spared sciatic nerve injury(SNI) as the experimental tool. Compared with the sham group, the SNI group exhibited diminished mechanical withdrawal threshold(MWT)(P<0.01), increased cold spray scores(P<0.01), glutamate(Glu), D-serine(D-Ser), and glycine(Gly) in extracellular fluids of ACC, and Glu, D-Ser, interleukin-1ß(IL-1ß), and lactic acid(Lac) in extracellular fluids of SDH(P<0.05), dwindled tumor necrosis factor(TNF-α)(P<0.05), and elevated protein levels of NMDAR-2 B, Src, and Panx1 in ACC(P<0.05). Compared with the SNI model rats, high-and medium-dose CRE(CRE-H/M) could potentiate the analgesic activity as revealed by the MWT test(P<0.05) and CRE-M enabled the decrease in cold spray scores(P<0.05). CRE-H/M could inhibit the levels of Glu, D-Ser and Gly in the extracellular fluids of ACC(P<0.05), and the levels of Glu in the extracellular fluids of SDH(P<0.05) in SNI rats. CRE-M significantly increased the levels of glucose(Gluc), Lac, interferon-gamma(IFN-γ), keratinocyte chemoattractant/human growth-regulated oncogenes(KC/GRO), and IL-4 in extracellular fluids of SDH in SNI rats(P<0.05). CRE-H/M/L could also inhibit the levels of NMDAR-2 B, Src and Panx1 in ACC and SDH in SNI rats(P<0.05). The central analgesic effect of CRE is presumedly related to the inhibited release of excitatory amino acid transmitters(Glu, D-Ser and Gly) in ACC and SDH of SNI rats, decreased protein expression of NMDAR-2 B, Src and Panx1 in the two regions, and the regulation of the Panx1-Src-NMDAR-2 B pathway in the spinal cord and brain. The above findings partially clarified the scientific basis of clinical analgesic effect of Chuanxiong Rhizoma.

[Shenlian extract attenuates TNF-α-induced ECV304 injury by regulating Nrf2/Keap1 signaling pathway].

This study aimed to investigate the effect and the possible mechanism of Shenlian( SL) extract on tumor necrosis factor-α( TNF-α)-induced ECV304 injury. After the establishment of TNF-α-induced ECV304 cells injure model,MTT assay was used to detect cell viability and the level of reactive oxygen species( ROS) was measured by flow cytometry. The contents of superoxide dismutase( SOD),malondialdehyde( MDA),nitric oxide( NO),endothelin-1( ET-1) and interleukin-1ß( IL-1ß) in the supernatant were detected by biochemical method and enzyme linked immunosorbent assay( ELISA). The expression levels of apoptosis-related proteins B-lymphoma-2 gene( Bcl-2),Bcl-2 associated X protein( Bax),caspase-3,caspase-9 and nuclear factor E2 associated factor2( Nrf2)/Kelch like epichlorohydrin associated protein-1( Keap1) signaling pathway related proteins Nrf2,Keap1,quinone oxidoreductase( NQO1) and heme oxygenase 1( HO-1) were detected by Western blot. The results showed that 50 µg·L-1 TNF-α significantly damaged ECV304 cells,induced the impairment of cell viability( P<0. 01),the increase of ROS production,the decrease of SOD activity,and the increase of MDA,NO,ET-1 and IL-1ß( P<0. 01),meanwhile,it caused the up-regulation of Keap1,caspase-9 and Bax protein expression,and down-regulation of NQO1 and Bcl-2 protein expression( P<0. 05) compared with the control group.Compared with the model group,SL extract reduced the damage of ECV304 cells induced by TNF-α,improved cell viability,reduced ROS production,increased SOD activity and decreased MDA,NO,ET-1,IL-1ß content( P<0. 01 or P<0. 05). In addition,SL extract also down-regulated the protein expression levels of Keap1,caspase-3,caspase-9 and Bax,and increased the protein expressions of Nrf2,NQO1,HO-1 and Bcl-2( P<0. 01 or P<0. 05). The above results indicate that SL extract can provide protective effect on ECV304 cells injury induced by TNF-α,alleviate oxidative stress injury,inflammation and apoptosis,and its mechanism may be related to regulating Nrf2/Keap1 signaling pathway.

Amelioration of muscular spasm-induced pain of Guangtongxiao recipe in a non-everted gut sac in vitro model.

BACKGROUND: The modern study of the traditional Chinese medicine (TCM) compound recipes is a complex issue because of the large number of components in the recipes that would produce several metabolites after entering the body. The TCM compound recipes are known to have the advantage of synergistic treatment by multiple targets due to diverse components. Therefore, a research method that can reflect the overall effect of compounds with multi-components is essential. The pharmacological studies of the classic TCM compound recipes mainly use the sero-pharmacological method. It is a semi-in vivo study method, and the drug to be tested in the in vitro experiment is the drug-containing serum from the model animals (the drug to be tested is a mixed drug system containing the prototype drugs in animal bodies that have pharmacodynamic effects and the metabolites). Herein, a safe and effective external TCM recipe was used to develop another semi-in vivo experimental method to reflect the overall effects of TCM. AIM: To observe the effects of in-vitro intestinal absorption liquid of aqueous extracts of the TCM compound recipe-Guangtongxiao foam aerosol (Guangtongxiao)-on the tension of isolated rectal rings of mouse and investigate the underlying mechanisms of antispasmodic and amelioration of muscular spasm-induced pain. METHODS: Intestinal absorption liquid of the Guangtongxiao aqueous extract at the five time points (30, 45, 75, 105, and 120 min) was prepared using a non-everted gut sac method. The isolated rectal rings of mice were prepared by pre-contraction using potassium chloride (KCl) or acetylcholine chloride (ACh) to make steady contraction. The intestinal absorption liquid were added cumulatively to the sink with the constricted rectal rings. The effects of the five groups of the intestinal absorption liquid with different drug concentration were observed on the tension of the isolated rectal rings. Then the ex vivo perfusion of the mouse rectal ring was performed as same as Guangtongxiao intestinal absorption liquid experiments, and the effects of two major components of Guangtongxiao, paeoniflorin (Pae) and tetrahydropalmatine (THP), on the rectal ring pre-treated with high concentration of KCl and ACh to induce contraction were studied. RESULTS: The relaxation rate of the five groups of the intestinal canals increased significantly with 3200 µL cumulative sample volume as compared to the blank group (P < 0.01). It suggested that the relaxation activity of the intestinal absorption liquid enhanced significantly with the prolongation of the interaction between isolated rectal rings and intestinal absorption liquid in a time-dependent manner. Also, significant differences were detected while comparing between the 120-min intestinal absorption liquid group and the blank group with respect to various cumulative sampling volumes (P < 0.01). In addition, the intestinal relaxation rate elevated gradually with the increase in sampling volume, indicating that the concentrations of active substances in the intestinal absorption liquid prepared by the non-everted gut sac model increased and the intestinal relaxation activity was enhanced with the prolongation of the absorption time in a dose-dependent manner. And Pae and THP in a concentration-dependent manner caused relaxation of the rectal ring, which is pretreated with high K+(KCl) and ACh to induce contraction. The EC50 of Pae and THP was 8.67 × 10-5 M (6.68 × 10-5-1.13 × 10-4) and 1.41 × 10-4 M (1.24 × 10-4-1.61 × 10-4) in the contraction model induced by KCl, and was 6.15 × 10-5 M (4.47 × 10-5-8.45 × 10-5), and 1.31 × 10-4 M(1.22 × 10-4-1.42 × 10-4) in the model induced by ACh, respectively. CONCLUSION: The intestinal absorption liquid of Guangtongxiao exerted a remarkable relaxation activity for the rectal rings, and relaxation of the smooth muscle tension might be one of the antispasmodic mechanisms of Guangtongxiao compound recipe. Also, adopting a semi-in-vivo experimental method to study the efficacy of topical external TCM recipe medicine is optimal.

[Screening combination ratio and exploring mechanism of Momordicae Semen and Epimedii Folium].

The aim of this paper was to obtain low toxicity and high efficiency anti-tumor Chinese medicine through screening the combination ratios of Momordicae Semen and Epimedii Folium, and to explore the anti-tumor mechanism of the combination of two drugs by observing their effect on apoptosis-related proteins in cancer cells. Methyl thiazolyl tetrazolium(MTT) assay was used to observe the effect of drug combination on the proliferation of tumor cells from different tissue sources. The effects of the combination of the two drugs on tumor cells were analyzed by Compusyn software. Plate cloning assay was used to observe the effect of combination of these two drugs on the proliferation of A549 cells in vitro. The expression of reactive oxygen species(ROS) and apoptotic proteins p53, Bcl-2 and Bax were compared by using ROS kit and Western blot. Lewis lung cancer model was used to observe the anti-tumor effect of drugs in vivo. The results showed that the anti-tumor effect of their ethanol extract was more significant than that of water extract, and the anti-proliferation effect was strongest when the ratio was 1∶1(P<0.05). Compusyn analysis showed that the combination of the two drugs had synergistic effect. Further studies showed that after combined use, the number of clonogen formation in A549 cells was significantly reduced(P<0.01); ROS production was increased; the expression of apoptosis-related protein p53 was up-regulated, and the ratio of Bcl-2/Bax was decreased. In vivo animal study showed that the tumor inhibition rate was 53.06%(P<0.05) in the high dose group. As compared with the single use of the two drugs, the combination of the two drugs had more significant anti-proliferative effect on tumors, and the optimum ratio was 1∶1. The combination of the two drugs at a ratio of 1∶1 inhibited the proliferation of various tumor cells, and had no significant effect on normal liver cells LO2 when compared with other ratios. Therefore, it can be preliminarily inferred that the combination of the two drugs may have the effect of synergism and detoxification. Further studies showed that the combination of the two drugs can significantly inhibit the proliferation of A549 cells, and its mechanism may be related to the activation of endogenous apoptotic pathway. In vivo experiments also showed that the tumor inhibition rate increased with the increase of drug concentration.

Extract of Caulis Spatholobi, a novel platelet inhibitor,efficiently suppresses metastasis of colorectal cancer by targeting tumor cell-induced platelet aggregation.

Tumor cell-induced platelet aggregation (TCIPA) is the core mechanism potentiating high viability for circulatory tumor cells,which is the rate-limiting factor for metastasis.Additionally,as supported by the successful application of aspirin,the pro-malignant effects during tumor-platelets interaction can be largely neutralized by pharmacological deactivation of platelets.Caulis Spatholobi is widely used as an anti-coagulation herb in traditional Chinese medicine,indicating its potential against TCIPA.In our study,three fractions of Caulis Spatholobi extracts were firstly prepared.In colorectal cancer(CRC) model,the anti-metastatic potential was evaluated both in vitro and in vivo followed by the detection of their platlet regulatory effects.Results showed that all three extracts significantly suppressed the invasion and metastasis of CRC.Mechanistically,by blocking platelet-derived PDGF-B releasing,they reversed the enhanced epithelial mesenchymal transition during MC38-platelets interation.Further,ethyl acetate fraction shows the most promising efficacy for the future application in treatment.Overall,our study have for the first time proved CaulisSpatholobi extracts,especially the ethyl acetate fraction,as a potent TCIPA inhibitor during metastatic progression,which provided a novel candidate for pharmacologically blockage of metastasis in CRC.

[Potential therapies for COVID-19 cardiovascular complications using artemisinin and its derivatives intervene based on its cardiovascular protection].

Corona virus disease 2019(COVID-19) has brought untold human sufferings and economic tragedy worldwide. It causes acute myocardial injury and chronic damage of cardiovascular system, which has attracted much attention from researchers. For the immediate strategy for COVID-19, "drug repurposing" is a new opportunity for developing drugs to fight COVID-19. Artemisinin and its derivatives have a wide range of pharmacological activities. Recent studies have shown that artemisinin has clear cardiovascular protective effects. This paper summarizes the research progress on the pathogenesis the pathogenesis of COVID-19 in cardiovascular damage by 2019 novel coronavirus(2019-nCoV) virus from myocardial cell injury directly by 2019-nCoV virus,viral ligands competitively bind to ACE2 and then reduce the protective effect of ACE2 on cardiovascular disease, "cytokine storm" related myocardial damage, arrhythmia and sudden cardiac death induced by the infection and stress, myocardial injury by hypoxemia, heart damage side effects from COVID-19 drugs and summarizing the cardiovascular protective effects of artemisinin and its derivatives have activities of anti-arrhythmia, anti-myocardial ischemia, anti-atherosclerosis and plaque stabilization. Then analyzed the possible multi-pathway intervention effects of artemisinin-based drugs on multiple complications of COVID-19 based on its specific immunomodulatory effects, protective effects of tissue and organ damage and broad-spectrum antiviral effect, to provide clues for the treatment of cardiovascular complications of COVID-19, and give a new basis for the therapy of COVID-19 through "drug repurposing".

[Research progress of effect of artemisinin family drugs on T lymphocytes immunomodulation].

Artemisinin was isolated from traditional Chinese herb Artemisia annua for treating malaria. A series of derivatives,like dihydroartemisinin,artesunate,artemether,artether,had the same core chemical structure,and sesquiterpene lactone containing peroxide bridge constitute the basic chemical structure. Besides anti-malaria,artemisinin family drugs were found to ameliorate many different diseases,which have attracted wide attention in recent years. Among different diseases,artemisinin family drugs were found to have T lymphocytes immunomodulation effects,including activation,proliferation,differentiation,apoptosis and subsets function. Because T cell immunologic response is the key point of many diseases,and impact the pathogenic process,therapeutic effect and prognosis,the drug studies with it as the target have become hotspots in recent years. Studies of artemisinin family drug on T cell immunomodulation were still at the initial stage and involved in different disease; furthermore,T cell immune process involves complicated molecular mechanism,it is imperative to summarize the advance of current studies for further systematic explanation and exploration of their characteristics and mechanisms. This article will summarize the research progress of artemisinin family drugs for malaria,autoimmune disease,hypersensitivity reaction,tumor,schistosomiasis and AIDS relating to T cell immune modulation,so as to provide basic and professional reference for related research and application.

[Efficacy and mechanism of Lianhua Qingwen Capsules(LHQW) on chemotaxis of macrophages in acute lung injury (ALI) animal model].

This paper was mainly to discuss the potential role and mechanism of Lianhua Qingwen Capsules(LHQW) in inhibiting pathological inflammation in the model of acute lung injury caused by bacterial infection. For in vitro study, the mRNA expression of MCP-1 in RAW264.7 cells and THP-1 cells, the content of MCP-1 in cell supernatant, as well as the effect of LHQW on chemotaxis of macrophages were detected. For in vivo study, mice were randomly divided into 7 groups, including normal group, model group(LPS 5 mg·kg~(-1)), LHQW 300, 600 and 1 200 mg·kg~(-1)(low, middle and high dose) groups, dexamethasone 5 mg·kg~(-1) group and penicillin-streptomycin group. Then, the anal temperature was detected two hours later. Dry weight and wet weight of lung tissues in mice were determined; TNF-α and MCP-1 levels in alveolar lavage fluid and MCP-1 in serum were detected. In addition, the infiltration of alveolar macrophages was also observed and the infiltration count of alveolar macrophages was measured by CCK-8 method. HE staining was also used to observe the inflammatory infiltration of lung tissues in mice. Both of the in vitro and in vivo data consistently have confirmed that: by down-regulating the expression of MCP-1, LHWQ could efficiently decrease the chemotaxis of monocytes toward the pulmonary infection foci, thus blocking the disease development in ALI animal model.

[Basic research of fibrosis on atherosclerotic plaque stability and related drug application].

In the background of the high incidence and high mortality of cardiovascular diseases,atherosclerosis is the main pathological feature of cardiovascular diseases and the core pathological basis for disease progression. In the evolution of atherosclerotic plaques,the rupture of unstable plaques,plaque shedding and formation of thrombosis are the most dangerous parts. In this process,the formation of plaque fibrosis is the core mechanism regulating plaque stability. Additionally,fibrosis reflects dynamic changes in the inflammatory processes and pathological changes. In view of the inflammation regulation and fibrosis regulation,this paper clarified the process of atherosclerotic plaque,explained the roles of relevant inflammatory cells and cytokines in plaque stability,and summed up drug researches related with stable plaque in recent years. In the future,improving the fibrosis will be a new idea for stabilizing plaque in atherosclerosis drug development.

[Research advancement in natural anti-cancer product].

Human health has been severely threatened by malignant tumors continuously.Rational and effective drug use provides an effective means for the treatment of malignant tumors,and is expected to become an important way to solve the problem of tumor treatment in the future.In recent years,with the escalation of new cancer theories and the emergence of clinical drug resistance,innovative research and development of anti-cancer drugs has always been a hot spot and focus in cancer research.Among them,the discovery of novel anti-cancer drugs from natural compound is of top priority due to its strong anti-cancer efficacy and the abundant drug resources.Therefore,it is imperative to systematically summarize the cutting-edge advancements of the natural products and their potential pharmacological mechanisms according to the characteristics of tumor progression,and put forward the new directions and trends for further development of anti-cancer natural products in the future.Specifically,the research advancements on anti-cancer effect of natural products were reviewed,focusing on both the traditional and innovative application.We hope this review could bring the light on the research path of the natural anti-cancer products clearly and comprehensively,and also provide inspirations for innovative,safer and more effective anti-cancer drug development and exploration.

[Research initiative of new thought on "main effect" of TCM formulae--new thinking on mechanism of compound action and compatibility mechanism of Chinese herbal compound formulae].

This article proposes a new thought on the study of "main effect" of traditional Chinese medicine (TCM) formulae. The blood concentrations of the pharmacodynamic substances of Chinese material medica（CMM）are usually very low, with lower toxic and side effects than western medicine. Therefore, according to a recent hypothesis of additive effect of multiple components for a single target, local targets in multi-component multi-target synergistic effect network of TCM may have the additive effect of similar components. Studies on the disposition of CMM showed that a constituent could bebio-transformed to many metabolites; these compounds with a similar structure are likely to have the same pharmacological effects on the same target, which could provide experimental evidences for the hypothesis of "additive effect". The authors of this article further believe that additive effect of TCM multi-components only comes up under a limited conditions/concentration. Because of the complexity of TCM-organism system, the complex effect of multicomponent addition and competition/antagonism is more likely to appear in single targets of drug effect. This complex effect may be the key to impact the synergistic effect of TCM multi-targets. In theory, choose and create a single target additive effect could realize the scientific compatibility of TCM and improve the curative effect and attenuate toxicity. According to the clinical demand and under the guidance of the above thought, we proposed the "main effect" of TCM formulae. Because traditional Chinese medicine (compounds) have diverse and complex effects, how to better study TCM formulae compatibility mechanism and improve the curative effect? Efforts shall be made to select one or several effects relating to clinical specific syndromes from the complex and diverse effects of TCM as the "main effect". The "main effect" of TCM formulae is the macroscopic manifestation of the synergistic effect of multi-component/multi-target. The study of the Formulae "main effect" can contain at least two aspects: one is the study of pharmacokinetic application of TCM formulae, and another is the study for pharmacodynamics effect. In the study of main effect, there are two main elements. First, which drug targets are directly related to the main effect? This requires identifying the target network. Second, which drug components positively or negatively control the single target of the target network? And what change in single target effect as well as the multi-target synergistic effect will be caused by the regulatory component concentration or the change in number? These two elements is the key to elucidate the mechanism of compound action and compatibility mechanism of Chinese herbal compound formulae. Through the study of the main effect, the clinical curative effect and the mechanism of the TCM formulae shall be improved.

[Effect of dihydroartemisinin on permeability of human erythrocyte membrane infected plasmodium].

In view of the fact that the antimalarial effects of artemisinins are significant but the mechanism has not yet been clarified and there are many different opinions, it is possible that artemisinins can produce high anti-malarial efficacy through various mechanisms and multiple pathways. In addition, the researches on the pathogenesis of malaria "erythrocyte membrane plasmodial surface anion channel (PSAC)" in the past few years have provided more positive findings, which may confirm and discover the new antimalarial mechanism of artemisinins. This paper was as to study the effect of dihydroartemisinin (DHA) in vitro on erythrocyte membrane permeability of HB3 plasmodium infection, with using the mechanism of 5% sorbitol can be used to kill the Plasmodium falciparum in red blood cell membrane selectively, the effectual difference of sorbitol on the killing of P. falciparum with adding DHA or not was detected, so as to investigate whether DHA can affect the permeability of the erythrocyte membrane. Result showed that, Pre-stimulation with 10 nmol·L⁻¹ DHA (the final concentration of plasmodium in vitro culture system) for 30 min could significantly decrease the killing effect of sorbitol on the HB3 plasmodium in the P. falciparum erythrocytic cycle, and DHA may inhibit the permeability of the erythrocyte membrane for preventing sorbitol through the red blood cell membrane, thereby reducing the killing effect of sorbitol on the P. falciparum.

Wuji Wan Formula Ameliorates Diarrhea and Disordered Colonic Motility in Post-inflammation Irritable Bowel Syndrome Rats by Modulating the Gut Microbiota.

Emerging evidence suggests that gut microbiota contribute to the treatment of post-inflammatory irritable bowel syndrome (PI-IBS). Our previous studies have demonstrated that a Chinese formula, Wuji Wan, has the ability to mitigate abdominal pain and diarrhea in PI-IBS rats. However, little is known about the underlying mechanism and whether the gut microbiota mediate the effect of Wuji Wan on PI-IBS. Thus, the aim of this study was to determine whether Wuji Wan mitigated PI-IBS by modifying the gut microbiota. PI-IBS was induced in Sprague-Dawley rats by enema using 4% acetic acid and restraint stress. Rats were fed water, Wuji Wan extract (630 mg/kg) or pinaverium bromide (13.5 mg/kg). Our data showed that Wuji Wan effectively ameliorated abdominal pain, colonic motility abnormality and visceral hypersensitivity. Analysis of the fecal microbiota showed that Wuji Wan could reverse the reduction in richness of the gut microbiota and significantly increase the relative abundances of Akkermansia, Bacteroides, and Parasutterella; however, Lactobacillus and Prevotella were markedly decreased in the PI-IBS rats. Moreover, Wuji Wan promoted goblet cell proliferation in the colonic mucosa by increasing the release of mucin, up-regulating the distribution of tight junction proteins Occludin and ZO-1 and down-regulating the expression of MLCK in colonic epithelial cells. These findings suggest that Wuji Wan may remit IBS by modulating the gut microbiota and stabilizing the gut mucosal barrier, indicating that the use of a classical formula of Traditional Chinese Medicine (TCM) that exhibits a prebiotic effect may be a promising strategy for PI-IBS treatment.