This study aims to establish the S_(180) tumor-bearing mice model, and to investigate the influence of Shenqi Erpi Granules(SQEPG) on immune function, as well as the drug's tumor-suppressive effect and mechanism. SPF grade KM mice(half male and half female) were randomly divided into 6 groups: a control group, a model group, a cyclophosphamide group(50 mg·kg~(-1)), as well as SQEPG groups in low-, medium-, and high-dose(5.25, 10.5, 21 g·kg~(-1)). The control group and the model group were given distilled water, and the other 4 groups were given the corresponding drugs by gavage. The administration continued for 10 days before the mice were sacrificed. The antitumor and immune regulation effects of SQEPG were evaluated. The effect of SQEPG on delayed type hypersensitivity reaction(DTH), carbon clearance index, and serum hemolysin antibody level was observed to reflect the effect on the immune function of tumor-bearing mice. Tumor weight was recorded to calculate the tumor suppression rate and the immune organ index. Hematoxylin-eosin(HE) staining was used to detect morphological changes in tumor tissues. Flow cytometry was employed to detect the percentage of CD4~+ and CD8~+ T-cells in the spleen tissues and the tumor tissue apoptosis levels. Immunohistochemistry was conducted to detect the KI67 protein expression level of tumor tissues. ELISA resorted to the detection of the following expression levels in tumor tissues: tumor necrosis factor-α(TNF-α), interleukin-2(IL-2), interferon-γ(IFN-γ). Western blot was performed to detect the expression levels of caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cyclin-dependent kinases 4(CDK4), G_1/S-specific cyclin D1(cyclin D1), and vascular endothelial growth factor A(VEGFA). The results showed that, compared with the model group, the SQEPG could increase the swelling of the auricle of the tumor-bearing mice; significantly increase the phagocytic index of carbon granule contour(P<0.05 or P<0.01), and the middle dose of SQEPG could significantly increase the antibody level of hemolysin(P<0.05); different doses of SQEPG significantly inhibit the growth of the tumor, and decrease the mass of the tumor tissues(P<0.05 or P<0.01); the low dose of SQEPG significantly decreased spleen index(P<0.05), low and high doses of SQEPG increased thymus index, while medium doses of SQEPG decreased thymus index. High doses of SQEPG significantly elevated the levels of CD4~+ and CD8~+ T-cells in the spleens of the homozygous mice(P<0.01 or P<0.001), and increased the apoptosis rate of the cells of the tumor tissues(P<0.05); Meanwhile, high-dose SQEPG elevated the levels of immunity factors such as IL-2, IFN-γ and TNF-α in the serum of tumor-bearing mice(P<0.01); medium-and high-dose SQEPG significantly lowered the rate of positive expression of KI67 protein in tumor tissues(P<0.01). Compared with the model group, high-dose SQEPG significantly up-regulated the expression of caspase-3 and Bax proteins in tumor tissues(P<0.05), and significantly down-regulated the expression of CDK4, cyclin D1, and VEGFA proteins(P<0.05 or P<0.01). In conclusion, SQEPG has the effect of improving immune function and inhibiting tumor growth in tumor-bearing mice. Its mechanism of tumor-suppressive effects may be related to apoptosis promotion, cell cycle progression block, and tumor cell proliferation inhibition.
Animals ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Male ; Female ; Apoptosis/drug effects* ; Sarcoma 180/genetics* ; Humans

AIM To optimize the extraction process for total polyphenols from Conioselinum vaginatu(Spreng.)Thell.,make component analysis,and evaluate their anti-oxidant,hypoglycemic activities.METHODS The effects of ultrasound,enzymatic hydrolysis,acid hydrolysis,alcohol extraction and hydrolysis processes on the extraction quantity of total polyphenols were investigated,respectively.With extraction temperature,extraction time,ethanol concetration and liquid-solid ratio as influencing factors,extraction quantity of total polyphenols as an evaluation index,the extraction process was optimized by response surface method.HPLC was adopted in the identification of polyphenolic composition and determination of their contents.Subsequently,total polyphenols' scavenging capacities on DPPH,ABTS,OH free radicals,total reducing power and inhibitory capacity on α-glucosidase were determined.RESULTS The highest extraction quantity of total polyphenols was observable when extraction process was employed.The optimal conditions were determined to be 62 ℃ for extraction temperature,54 min for extraction time,69％for ethanol concentration,and 50∶1 for liquid-solid ratio,the extraction quantity of total polyphenols was(9.51±0.2)mg GAE/g.Seven constituents existed in C.vaginatu,among which ferulic acid demonstrated the highest content,followed by that of myricetin,while D-tryptophan content was the lowest.At the concentration of 7.61 mg/L,total polyphenols displayed the scavenging rates on DPPH,ABTS,OH free radicals of 80.70％,85.97％,28.60％,total reducing power of 0.22,and inhibition rate on α-glucosidase of 77.23％,respectively.CONCLUSION This stable and reliable method can be used for the extraction of total polyphenols from C.vaginatum with strong anti-oxidant,hypoglycemic activities.

AIM To optimize the extraction process for total polyphenols from Conioselinum vaginatu(Spreng.)Thell.,make component analysis,and evaluate their anti-oxidant,hypoglycemic activities.METHODS The effects of ultrasound,enzymatic hydrolysis,acid hydrolysis,alcohol extraction and hydrolysis processes on the extraction quantity of total polyphenols were investigated,respectively.With extraction temperature,extraction time,ethanol concetration and liquid-solid ratio as influencing factors,extraction quantity of total polyphenols as an evaluation index,the extraction process was optimized by response surface method.HPLC was adopted in the identification of polyphenolic composition and determination of their contents.Subsequently,total polyphenols' scavenging capacities on DPPH,ABTS,OH free radicals,total reducing power and inhibitory capacity on α-glucosidase were determined.RESULTS The highest extraction quantity of total polyphenols was observable when extraction process was employed.The optimal conditions were determined to be 62 ℃ for extraction temperature,54 min for extraction time,69％for ethanol concentration,and 50∶1 for liquid-solid ratio,the extraction quantity of total polyphenols was(9.51±0.2)mg GAE/g.Seven constituents existed in C.vaginatu,among which ferulic acid demonstrated the highest content,followed by that of myricetin,while D-tryptophan content was the lowest.At the concentration of 7.61 mg/L,total polyphenols displayed the scavenging rates on DPPH,ABTS,OH free radicals of 80.70％,85.97％,28.60％,total reducing power of 0.22,and inhibition rate on α-glucosidase of 77.23％,respectively.CONCLUSION This stable and reliable method can be used for the extraction of total polyphenols from C.vaginatum with strong anti-oxidant,hypoglycemic activities.

Aim To study the permeability of salidro-side(Sal)to the blood brain barrier(BBB)by high-performance liquid chromatography electrospray ioniza-tion tandem mass spectrometry(UPLC-ESI-MS-MS),and to explore the target and mechanism of Sal in the treatment of ischemic stroke(IS)by network pharma-cology,molecular docking technique and animal exper-iment.Methods UPLC-ESI-MS/MS was used to study the BBB penetration of Sal.Multiple databases were used to predict the target of Sal and the disease target of IS,GO and KEGG enrichment analysis were performed and verified by molecular docking technique and animal experiments.Results After Sal adminis-tration to normal rats and MCAO rats,Sal prototype and the metabolite tyrosol were detected in plasma and brain tissue of rats.A total of 191 targets were identi-fied by network pharmacology,the enrichment analysis of GO mainly involved in the biological processes of proteolysis and positive regulation of cell migration,and the analysis of KEGG pathway suggested that PI3K-Akt,MAPK,FOXO and other signaling path-ways played a key role in the treatment of IS by Sal The results of molecular docking showed that Sal had good binding ability with the core target of docking,and the results of animal experiments showed that Sal could significantly improve the neurologic impairment of MCAO rats,the number of Nissl-positive cells in is-chemic side significantly increased,and the expression of VEGF,EGFR and IGF1 increased,while the ex-pression of IL-6 and MMP9 was inhibited.Conclu-sions Sal is able to penetrate the BBB and enter the central nervous system for its pharmacological effects.Network pharmacology predicts the core targets of Sal in the treatment of IS,including VEGFA,EGFR,IL-6,MMP9,IGF1,CASP3,ALB,SRC.The effects of Sal on some core targets can be verified by animal ex-periments,to provide a reference for further study of the mechanism of Sal in the treatment of IS.

Aim To study the neuroprotective effects of Herba siegesbeckiae extract on cerebral ischemia/ reperfusion rats and its mechanism. Methods Sixty SD rats were randomly divided into model group, low, middle and high dose groups of Herba siegesbeckiae, and Sham operation group, and the drug was given continuously for seven days. The degree of neurologic impairment was evaluated by mNSS, and the infarct volume was measured by MRI. The number of Nissl-posi- tive cells was detected by Nissl staining, and the apop- tosis was accessed by Tunel staining. Furthermore, the expression of Bax, Bcl-2 and NeuN was observed by Western blot, and the expression of NeuN was detected by immunofluorescence staining. The expression of IL- 1β, TNF-α and IL-6 mRNA was performed by RT- qPCR. Results The mNSS score and the volume of ischemic cerebral infarction in the model group were significantly increased, and Herba siegesbeckiae extract treatment significantly decreased the mNSS score and infarct volume (P<0.05, P<0.01). Herba siegesbeckiae extract could increase the number of Nissl-pos- itive cells and the expression of NeuN (P<0.01), and reduce the number of Tunel-positive cells (P<0.01). Western blot showed that Herba siegesbeckiae extract inhibited the expression of Bax, increased Bcl-2 and NeuN in ischemic brain tissue (P<0.01). RT-qPCR showed that Herba siegesbeckiae extract inhibited the expression of IL-1 β, TNF-α and IL-6 mRNA in the is-chemic brain tissue (P<0.01). Conclusions Herba siegesbeckiae extract can reduce the cerebral infarction volume, improve the neurological function damage, inhibit the apoptosis of nerve cells and the expression of inflammatory factors and promote the expression of NeuN, there by exerting protective effects on MCAO rats.

Objective To explore the effect of shikonin on the recovery of nerve function after acute spinal cord injury(SCI)in rats.Methods 96 male Sprague-Dawley(SD)rats were divided into 4 groups randomly:sham operation group(Group A),sham operation+shikonin group(Group B),SCI+DMSO(Group C),SCI+shikonin group(Group D).The acute SCI model of rats was made by clamp method in groups C and D.After subdural catheterization,no drug was given in group A.rats in groups B and D were injected with 100 mg·kg-1 of shikonin through catheter 30 min after modeling,and rats in group C were given with the same amount of DMSO,once a day until the time point of collection tissue.Basso-Beattie-Bresnahan(BBB)scores were performed on 8 rats in each group at 6,12,and 3 d after moneling,and oblique plate tests were performed on 1,3,7 and 14 d after modeling,and then spinal cord tissues were collected.Eight rats were intraperitoneally injected with propidine iodide(PI)1 h before sacrificed to detection PI positive cells at 24 h in each group.Eight rats were sacrificed in each group at 24 h after modeling,the spinal cord injury was observed by HE staining.The Nissl staining was used to observe survivor number of nerve cells.Western-blot technique was used to detect the expression levels of Bcl-2 protein and apoptosis related protein RIPK1.Results After modeling,BBB scores were normal in group A and B,but in group C and D were significantly higher than those in group A and B.And the scores in group D were higher than those in group C in each time point(P＜0.05).At 12 h after modeling,the PI red stained cells in group D were significantly reduced compared with that in group C,and the disintegration of neurons was alleviated(P＜0.05).HE and Nissl staining showed nerve cells with normal morphology in group A and B at 24h after operation.The degree of SCI and the number of neuronal survival in group D were better than those in group C,the differ-ence was statistically significant at 24h(P＜0.05).The expression of Bcl-2 and RIPK1 proteins was very low in group A and B;The expression of RIPK1 was significantly increased in Group C and decreased in Group D,with a statistically significant dif-ference(P＜0.05);The expression of Bcl-2 protein in group D was significantly higher than that in group C(P＜0.05).Conclu-sion Shikonin can alleviate the pathological changes after acute SCI in rats,improve the behavioral score,and promote the re-covery of spinal nerve function.The specific mechanism may be related to the inhibition of TNFR/RIPK1 signaling pathway mediated necrotic apoptosis.

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.
Humans ; Carcinoma, Hepatocellular/genetics* ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Liver Neoplasms/genetics* ; Molecular Docking Simulation ; Sincalide/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Hep G2 Cells ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis ; Autophagy ; Cell Proliferation ; Cell Line, Tumor ; STAT3 Transcription Factor/metabolism*

Artemisiae Argyi Folium is commonly used in clinical practice. Artemisiae Verlotori Folium, the dried leaves of Artemisia verlotorum, is often used as a folk substitute for Artemisiae Argyi Folium in Lingnan area. In this study, gas chromatography-triple quadrupole mass spectrometry(GC-MS) was used to detect the volatile oil components of 27 samples of Artemisiae Verlotori Folium and 13 samples of Artemisiae Argyi Folium, and the volatile components were compared between the two species. The internal standard method was combined with multi-reaction monitoring mode(MRM) to determine the content of six major volatile components. Hierarchical clustering analysis(HCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were carried out for the content data. The results showed that the Artemisiae Argyi Folium samples had higher content and more abundant volatile oils than the Artemisiae Verlotori Folium samples. Artemisiae Argyi Folium mainly had the components with lower boiling points, while Artemisiae Verlotori Folium mainly had the components with higher boiling points. Terpenoids were the main volatile components in Artemisiae Verlotori Folium(mainly sesquiterpenoids) and Artemisiae Argyi Folium(monoterpenoids). In addition, Artemisiae Argyi Folium had higher content of oxygen-containing derivatives than Artemisiae Verlotori Folium. Furthermore, the stoichiometric analysis showed that the two species could be distinguished by both HCA and OPLS-DA, indicating that the volatile components of the two were significantly different. This study can provide a scientific basis for the quality evaluation and data support for the local rational application of Artemisiae Verlotori Folium in Lingnan.
Gas Chromatography-Mass Spectrometry ; Chemometrics ; Oils, Volatile ; Drugs, Chinese Herbal ; Plant Leaves ; Artemisia

Aim To develop a ultra-high performance liquid chromatography electrospray-ionization tandem mass spectrometry ( UPLC-MS/MS ) method for the simultaneous determination of salidroside derivative pOBz in rat plasma and brain tissue, and to study the pharmacokinetic profile and penetration of the blood-brain barrier in rats after a single dose intravenous administration of pOBz. Methods SD rats were administered pOBz at a dose of 50 mg • kg