Rapid identification of the compounds of Bushen Huoxue Prescription based on offline two-dimensional liquid chromatography with high-resolution mass spectrometry and molecular network technology.

The comprehensive and efficient characterization of components in traditional Chinese medicine is crucial for elucidating its active constituents and uncovering its mechanism. Identifying the compounds of the Bushen Huoxue Prescription (BHP) is difficult because of its complex composition and the large difference in concentration among its compounds. In this study, a hydrophilic interaction liquid chromatography coupled with reversed-phase LC (HILIC × RPLC) offline 2D-LC tandem high-resolution mass spectrometry method was established to analyze the total compounds of the BHP. Database screening and molecular networking were performed to identify the compounds. In contrast to conventional 1D chromatography, 2D chromatography increased peak capacity, enriched trace ingredients, and prevented the masking of high-abundance compounds. A total of 165 compounds were identified, and 14 potential compounds needed to be further identified. This study provided an effective method for comprehensively analyzing the complex system of traditional Chinese medicine compounds.

Pharmacological mechanism and clinical study of Qiming granules in treating diabetic retinopathy based on network pharmacology and literature review.

ETHNOPHARMACOLOGY RELEVANCE: Diabetic retinopathy (DR) is a general complication of diabetes, which has become a serious threat to human health worldwide. However, the best treatment is still under development. Qiming (QM) granules are mainly composed of Astragalus membranaceus, Pueraria lobata, Rehmannia Glutinosa, Lycium barbarum, Cassiae Semen, Fructus Leonuri, Pollen Typhae, and Leech, which are beneficial to qi, nourish liver and kidney, and clear collaterals and eyes. The treatment of DR is a comprehensive application based on the above traditional Chinese medicine. In recent years, satisfactory results have been achieved for DR. AIM OF THE STUDY: Through the traditional application analysis, network pharmacology analysis and clinical research summary of QM granules, to review the effectiveness and advantages of QM granules in the treatment of DR comprehensively. MATERIALS AND METHODS: Analysis of main active components of QM granules by network pharmacology and prediction of mechanism of action of QM granules on DR. Further, PubMed, Web of Science, and China National Knowledge Infrastructure were used to search literature, using the keywords of "Qiming granules", "diabetic retinopathy", "clinical research" and their combinations, mainly from 1999 to 2022. RESULTS: Traditional pharmacological analysis, Network pharmacological analysis, animal experiments, and clinical studies have confirmed that QM granules plays a role in the treatment of DR through multiple components, multiple targets, and multiple channels. CONCLUSIONS: This systematic review for the first time provides meaningful information for the further study of the pharmacodynamic material basis and pharmacological mechanism of QM granules, and also provides a basis for further study of quality markers and quality control of QM granules.

Fangchinoline exerts anticancer effects on colorectal cancer by inducing autophagy via regulation AMPK/mTOR/ULK1 pathway.

Autophagy has become a promising target for cancer therapy. Fangchinoline (Fan) has been shown to exert anticancer effects in some types of cancers. However, the anticancer effects on colorectal cancer (CRC) and the underlying mechanisms have never been elucidated. More specifically, regulation of autophagy in CRC by Fan has never been reported before. In the present study, Fan was found to induce apoptosis and autophagic flux in the CRC cell lines HT29 and HCT116, which was reflected by the enhanced levels of LC3-II protein and p62 degradation, and the increased formation of autophagosomes and puncta formation by LC3-II. Meanwhile, combination with the early-stage autophagy inhibitor 3-methyladenine (3-MA) but not the late-stage autophagy inhibitor chloroquine (CQ) further increased Fan-induced cell death, which suggested the cytoprotective function of autophagy induced by Fan in both HT29 and HCT116 cells. Moreover, Fan treatment demonstrated a dose- and time-dependently increase in the phosphorylation of AMPK and decrease in the phosphorylation of mammalian target of rapamycin (mTOR) and ULK1, leading to the activation of the AMPK/mTOR/ULK1 signaling pathway. Furthermore, in the HT29 xenograft model, Fan inhibited tumor growth in vivo. These results indicate that Fan inhibited CRC cell growth both in vitro and in vivo and revealed a new molecular mechanism involved in the anticancer effect of Fan on CRC, suggesting that Fan is a potent autophagy inducer and might be a promising anticancer agent.

[Study on molecular mechanism of Solanum nigrum in treatment of hepatocarcinoma based on network pharmacology and molecular docking].

This study aimed to explore the main active ingredients and potential targets of Solanum nigrum(SN), so as to reveal the potential molecular mechanism of SN in the treatment of hepatocellular carcinoma(HCC) based on network pharmacology and molecular docking. First,the main active ingredients and predictive targets of SN were collected in the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP). Then,the targets relating to HCC were collected through retrieval of integrated bio-pharmacological network database for traditional Korean medicine(PharmDB-K), oncogenomic database of hepatocellular carcinoma(OncoDB.hcc). The common targets of disease-drug component were selected through intersection between predictive targets and disease targets. Next, based on the String platform, protein-protein interaction network(PPI) model of the potential anti-HCC targets was constructed using the software Cytoscape 3.7.1. ClueGO and CluePedia APP in Cytoscape were used to analyze the gene function of SN in the treatment of HCC, and construct the main active ingredients-potential targets-signal pathways topology network of SN. Finally,DISCOVERY STUDIO software was applied in verifying the molecular docking between the key active ingredient and potential protein target. The results showed that there were 4 main active ingredients of SN, involving 22 potential targets relating to HCC and 7 signal pathways relating to potential anti-HCC targets of SN. Network analysis showed that SN may play a therapeutic role in HCC by acting on key targets, such as EGFR, TP53, MYC, CCND1 and CTNNB1. Molecular docking results showed that quercetin and EGFR could bind stably and interact through amino acid residues LEU718, LYS745 and GLN791. This study revealed the potential active ingredients and the possible molecular mechanism of SN for treatment of HCC, providing scientific basis for follow-up exploration of the molecular mechanism of SN against HCC.

[Effect of electroacupuncture serum on expression of myogenic differentiation antigen and autophagy-related protein Beclin 1 in cultured muscle satellite cells under starvation].

OBJECTIVE: To explore the effect of electroacupuncture (EA) serum on expression of myogenic differentiation antigen (Myod) and autophagy-related protein Beclin 1 in cultured muscle satellite cells of rats under starvation conditions. METHODS: The primary multifidus muscle satellite cells of one male SD rat were isolated and cultured to obtain the 3rd generation of cells. The EA serum was got from the rat received EA stimulation of bilateral "Weizhong" (BL40, 2 Hz/10 Hz, 1 mA, duration of 20 min, once daily for 7 days). The cell suspension (2×104/well) of the 3rd generation of cultured cells was transferred to each well of a 96-well plate in medium containing 10% fetal bovine serum (FBS). Twelve duplicate wells were set up for the blank control serum (without FBS), 10% FBS, 10% EA serum, 20% EA serum and 30% EA serum groups and incubated for 12 h and 24 h, respectively. Each well was supplemented with 10 µL CCK-8 reagent to be incubated for 1 h again for observing the state of cell proliferation. After culturing the primary muscle satellite cells in serum-free medium for 12 h, the cells were randomly divided into serum-free group, 10% fetal bovine serum group and optimal concentration electroacupuncture serum group, and serum of corresponding concentration was added respectively. The expression levels of Beclin 1 and cell-proliferation-related protein Myod were detected by Western blot. RESULTS: CCK-8 assay displayed that the proliferation levels were significantly higher at 12 h and 24 h after serum intervention in the 10% FBS, 10% EA serum, 20% EA serum and 30% EA serum groups than that in the blank control serum group (P<0.01), and at 24 h in the 3 EA serum groups than in the 10% FBS group (P<0.01), but without significant difference among the three EA serum groups (P>0.05). As a result, 10% EA serum was selected as the optimal concentration for Western blot tests. No significant difference was found in the expression levels of Myod and Beclin 1 proteins among the serum-free, 10% FBS and 10% EA serum groups before intervention (P>0.05), and there was a marked up-regulation of Myod expression and an obvious down-regulation of Beclin 1 expression at 12 h in both the 10% EA serum and 10% FBS groups in comparison with their own pre-intervention (P<0.05). There were a marked up-re-gulation of Myod expression at both 12 h and 24 h and Beclin 1 expression at 24 h in both the 10% EA serum group and 10% FBS group than that in the serum-free group (P<0.05), and an obvious down-regulation of Beclin 1 expression at 12 h in both 10% FBS and 10% EA serum groups than that in the serum-free group (P<0.05, P<0.01). After 24 h's serum intervention, there was an increase of Myod expression and a reduction of Beclin 1 expression in both 10% FBS and 10% EA serum groups compared with those after the 12 h intervention (P<0.05). No significant differences were found between the 10% FBS and 10% EA serum groups in the expression levels of Myod and Beclin 1 proteins (P>0.05). CONCLUSION: EA serum can promote proliferation of cultured muscle satellite cells under starvation conditions, which is related to its functions in regulating expression of Beclin 1 and cell-proliferation-related protein Myod.

[Clinical trials of treatment of post-stroke foot drop by fire-needling plus stuck-needle-stretching].

OBJECTIVE: To compare the therapeutic effect of fire-needling plus stuck-needle-stretching and conventional acupuncture for post-stroke foot drop, so as to provide a reference foundation for optimizing clinical treatment regimen. METHODS: A total of 60 patients with post-stroke foot drop were equally randomized into a treatment group and a control group. In the treatment group, fire-needling plus stuck-needle-stretching was applied to acupoint pairs Jiexi (ST41)-Taichong (LR3), Xiajuxu (ST39)-ST41, Fenglong (ST40)-ST39, shangjuxu (ST37)-ST40, Zusanli (ST36)-ST37, Dubi (ST35)-ST36, Xuanzhong (GB39)-Qiuxu (GB40) on the affected side, and Chengshan (BL57) and Ashi points were swiftly pricked with red-hot filiform needle without needle retaining. In the conventional acupuncture group, the same acupoints were needled with filiform needles, and the treatment for both groups was given once a day, 6 times a week, for consecutive 4 weeks. The spasm severity of posterior triceps of the lower leg was evaluated using modified Ashworth scale, the tibial anterior muscle strength was measured using Lovett' and Martin's methods, the activities of daily living were assessed using modified Barthel's index, the walking ability was evaluated using Holden walking rating scale and the lower limb motor function assessed using Fugl-Meyer assessment scale, and the severity of foot drop was assessed in reference to Garceau criteria. RESULTS: After the treatment, the score of the modified Ashworth scale was significantly reduced (P<0.01), and those of Lovett muscle strength scale, Barthel index, Holden walking rating scale and Fugl-Meyer lower limb motor function scale were considerably increased in both groups versus their own pre-treatment (P<0.01). The therapeutic effect of the treatment group was significantly superior to that of the control group in reducing Ashworth scale score and in increasing the scores of Lovett muscle strength test, Barthel index, Holden walking function scale and Fugl-Meyer lower limb motor function scale (P<0.01). Of the two 30 cases in the control and treatment groups, 10(33.33%)and 14(46.67%) experienced a remarkable improvement, 10(33.33%)and 12(40.00%)were improved, 7(23.33%)and 3(10.00%)had a mild progress, 3(10.00%) and 1(3.33%) failed, with the excellent plus good effective rates being 66.67% and 86.67%, respectively, but without significant difference between the two groups (P>0.05). CONCLUSION: Both fire-needling plus stuck-needle-stretching and conventional acupuncture are effective in the treatment of post-stroke foot drop, but the former is relatively better.

In a Rat Model of Acute Liver Failure, Icaritin Improved the Therapeutic Effect of Mesenchymal Stem Cells by Activation of the Hepatocyte Growth Factor/c-Met Pathway.

Acute liver failure (ALF) is a serious life-threatening condition. Mesenchymal stem cells (MSCs) may be an effective treatment for this condition and a good alternative to liver transplantation. Icaritin (ICT) is an active ingredient of the genus Epimedium, a traditional Chinese medicine, with the potential to enhance the proliferation of MSCs. The purpose of this study was to explore whether ICT increased the therapeutic effects of MSCs and explore its underlying mechanisms. For in vivo experiments, a rat ALF model was established by intraperitoneal injection of D(+)-galactosamine/ lipopolysaccharide. MSCs cocultured with ICT were used to treat ALF rats and the protective effects assessed as survival rate, levels of serum AST and ALT, and histological changes in liver tissue. For in vitro experiments, MSCs were treated in serum-free culture for 72 h to simulate the disruption of intrahepatic microcirculation. MSCs apoptosis was examined to determine whether ICT rescued impaired MSCs. The role of the hepatocyte growth factor (HGF)/c-Met pathway in MSCs was assessed by constructing genetically modified MSCs overexpressing c-Met and by using the c-Met receptor inhibitor (crizotinib). The results showed that MSCs increased the survival rate of ALF rats and reduced liver damage. MSCs cocultured with ICT exerted a greater therapeutic effect than MSCs alone. Further, the HGF/c-Met pathway played a key role in the antiapoptotic activity of MSCs, which was associated with the optimized efficacy of ICT. In conclusion, this study demonstrated that ICT enhances the therapeutic effect of MSCs in a model of ALF, improving the antiapoptotic potential of MSCs by upregulation of the HGF/c-Met pathway. The combination of stem cell therapy with traditional herbal extracts may improve MSC-based clinical applications.

[Protective Effects of Cornus Officinalis Total Glycosides and Cornus Polysaccharides on Myocardial Mitochondria of Acute Myocardial Infarction Rats: an Experimental Study].

OBJECTIVE: To observe the effect of Cornus Officinalis total glycosides (COTG) and Cornus polysaccharides (CP) on myocardial mitochondria and expression levels of glycogen synthase kinase-3ß (GSK-3ß) of acute myocardial infarction (AMI) rats. METHODS: The AMI rat model was established by ligating the left anterior descending branch of coronary artery. Rats were divided into 5 groups according to random digit table, i.e., the sham-operation group, the model group, the COTG prevention group, the CP treatment group, the COTG treatment group, 12 in each group. Normal saline was administered to rats in the normal control group and the model group by gastrogavage. Corresponding medication was respectively administered to rats in the rest 3 groups by gastrogavage. The cardiac function was detected by echocardiography and hemodynamics. The infarct size was determined by Masson trichrome staining. The expression of mitochondrial biogenesis genes such as a subunit of peroxisome proliferators-activated receptor-γ coactivator-1 (PGC-1α), PGC-1ß, nuclear respiratory factor-1 (NRF-1), and GSK-3P mRNA were detected by Real-time PCR. RESULTS: Compared with the sham-operation group, the myocardial infarction size increased, cardiac function decreased, the expression of PGC-1α, PGC-1ß, and NRF-1 mRNA decreased, and the expression of GSK-3ß mRNA increased (all P <0. 05). Compared with the model group, myocardial infarction sizes were reduced, cardiac function was improved, the expression of NRF-1 mRNA was elevated in the COTG prevention group, the CP treatment group, the COTG treatment group; the expression of the PGC-1α and PGC-1ß mRNA was elevated in the COTG prevention group and the CP treatment group; the expression of GSK-3ß mRNA was reduced in the CP treatment group (all P <0. 05). Compared with the CP prevention group, fractional shortening (FS) and aortic systolic blood pressure (SBP) increased in the CP treatment group; ejection fraction (EF) decreased in the CP treatment group; the expression of PGC-1α, PGC-1ß, NRF-1 mRNA were reduced in the the CP treatment group and the COTG treatment group; the expression of GSK-3ß mRNA decreased in the CP treatment group (all P <0. 05). Compared with the COTG treatment group, FS, EF, left ventricular end systolic pressure (LVESP), SBP, and the expression of GSK-3ß mRNA were reduced in the CP treatment group (P <0. 05). CONCLUSIONS: COTG and CP could improve cardiac function, reduce the myocardial infarction area, and promote biogenesis of myocardial mitochondria. Their protective effects on the mitochondria of cadiocytes might be achieved by GSK-3ß signalina pathway.

[Inhibition of Jumi extraction on growth of human cervical cancer cell line HeLa].

OBJECTIVE: To explore the inhibition of Jumi (traditional Chinese medicine) extraction on the growth of human cervical cancer cell line HeLa. METHODS: Nude mouse model of human cervical cancer HeLa cell transplantation was established. The nude mice bearing cancer were randomly divided into control group and Jumi treated groups with different concentration (0.001, 0.002, 0.005, 0.01 mg/ml). The growth of cervical cancer cell in experimental mice were measured. Cultured HeLa cells were incubated in culture media with or without Jumi extract for 48 hours. Cell proliferation rate, cell apoptosis, caspase-3/7 and caspase-6 activity were determined by MTT colorimetric assay, flow cytometry analysis and spectrophotometric detection, respectively. RESULTS: With the increase of the concentration of Jumi extract, tumor-bearing mice tumor inhibition rate gradually increased. The proliferation of cultured HeLa cells were significantly inhibited by Jumi extract in a dose-dependent manner. IC50 was 0.004 mg/ml. Apoptosis rates in the cells treated with Jumi extract were higher than those of the control group. Compared with the control group, except for lower Jumi treated group (0.001 mg/ml), caspase-3/7 and caspase-6 activity were significantly increased in the all Jumi treated groups. CONCLUSION: Jumi extract can inhibit the proliferation of human cervical cancer cell line HeLa in vitro in a dose-dependent manner and promote cell apoptosis through caspase-3, caspase-7 and caspase-6 pathway.