[Mechanism of ursolic acid in regulating colorectal cancer cell HCT116 autophagy through hedgehog signaling pathway].

To prove that ursolic acid(UA)could activate the autophagy of colorectal cancer HCT116 cells by inhibiting hedgehog signaling pathway. The effect of UA on the viability of HCT116 cells was determined by MTT assay. The effect of UA on the proliferation and migration of HCT116 cells was detected by crystal violet staining and scratch test. In the study on autophagy, the time points were screened out first: the autophagy fluorescence intensity of UA acting on HCT116 at different time points were detected by Cell Meter~(TM) Autophagy Assay Kit; Western blot was used to detect the expression of autophagy protein P62 at different time points. Then, Cell Meter~(TM) Autophagy Assay Kit was used to detect the effect of UA on autophagy fluorescence intensity of HCT116 cells. The effect of different doses of UA on the expressions of LC3Ⅱ and P62 proteins in HCT116 cells were detected by Western blot. Further, AdPlus-mCherry-GFP-LC3 B adenovirus transfection was used to detect the effects of UA on autophagy flux of HCT116 cells; UA combined with autophagy inhibitor chloroquine(CQ) was used to detect the expression of LC3Ⅱ by Western blot. In terms of mechanism, the effect of UA on hedgehog signaling pathway-related proteins in HCT116 cells was detected by Western blot. The results showed that UA inhibited the activity, proliferation and migration of HCT116 cells. UA enhanced the fluorescence intensity of autophagy in HCT116 cells, while promoting the expression of LC3Ⅱ and inhibiting the expression of P62, in a time and dose dependent manner. UA activated the autophagy in HCT116 cells, which manifested that UA resulted in the accumulation of fluorescence spots and strengthened the fluorescence intensity of autophagosomes; compared with UA alone, UA combined with autophagy inhibitor CQ promoted the expression of LC3Ⅱ. UA reduced the expressions of PTCH1, GLI1, SMO, SHH and c-Myc in hedgehog signaling pathway, while increased the expression of Sufu. In conclusion, our study showed that UA activated autophagy in colorectal cancer HCT116 cells, which was related to the mechanism in inhibiting hedgehog signaling pathway activity.

[Inhibition of scutellarin on differentiation of colonic cancer stem cells via hedgehog signaling pathway].

The objective of this study was to investigate the inhibitory effect of scutellarin on the differentiation of colonic cancer stem cells in vitro and in vivo and to explore its underlying hedgehog signaling-based mechanism. The effect of scutellarin on the growth in vitro of HT-29 cells-derived cancer stem-like cells(HT-29 CSC) was observed with 3 D cell culture. The effect of scutellarin on the transformation of HT-29 CSC cells was assessed by soft agar colony formation assay. Fetal calf serum was used to induce differentiation of stem cells and observe the effect of scutellarin on HT-29 CSC cells differentiation in vitro. The effects of scutellarin on mRNA expressions of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells were determined by quantitative Real-time polymerase chain reaction(qRT-PCR). The effects of scutellarin on protein expressions of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells were examined by Western blot. After subcutaneous implantation of HT-29 CSC cells in nude mice, the effect of scutellarin on the mouse body weight and the growth of HT-29 CSC-derived tumor were explored. qRT-PCR was used for evaluating the effect of scutellarin on mRNA levels of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog in tumor. Western blot and immunohistochemistry analysis were used to detect the effect of scutellarin on protein expressions of c-Myc, Gli1, Lgr5, CD133 and Ki-67 in tumor. The in vitro experiments showed that scutellarin inhibited the growth, transformation and differentiation of HT-29 CSC cells, significantly down-regulated the mRNA levels of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells as well as the protein expression levels of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells. Additionally, animal experiments showed that scutellarin significantly inhibited the growth of subcutaneous xenografts in nude mice, and down-regulated the mRNA expressions of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog as well as the protein levels of c-Myc, Gli1, Lgr5, CD133 and Ki-67 of xenografts in nude mice. Taken together, scutellarin could inhibit the differentiation of colo-nic cancer stem cells in vitro and in vivo, potentially by down regulation of hedgehog signaling pathway activity.

Identification of the Active Compounds and Significant Pathways of Artemisia Annua in the Treatment of Non-Small Cell Lung Carcinoma based on Network Pharmacology.

BACKGROUND Artemisia annua exerts powerful effects in non-small cell lung carcinoma (NSCLC). Some studies have shown that Artemisia annua possesses the characteristics of new therapeutic drugs for NSCLC patients. However, the underlying molecular mechanism of Artemisia annua anti-NSCLC is not yet fully elucidated because Artemisia annua contains hundreds of ingredients. This study aimed to conduct network pharmacological analysis on the mechanism of action of Artemisia annua against NSCLC. MATERIAL AND METHODS The active ingredients and corresponding potential targets of Artemisia annua were searched and screened in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Then through The Cancer Genome Atlas (TCGA) and the National Center for Biotechnology Information (NCBI) databases to establish NSCLC related targets. Based on the matching results of Artemisia annua potential targets and NSCLC targets, a protein-protein interaction (PPI) network was constructed to analyze the interactions between these targets and topologically screen the central targets. Furthermore, Gene Ontology (GO) biological functions analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathways enrichment were carried out. RESULTS There were 19 main active ingredients of Artemisia annua screened for target prediction; 40 NSCLC-related common targets were identified via multiple NSCLC databases. The node area and corresponding degree value of AKT1, MYC, CCND1, VEGFA, JUN, MAPK1, EGFR, and ESR1 were large and could be easily found in the PPI network. The aforementioned results were further verified by the analysis of GO biological function and KEGG enrichment analysis. CONCLUSIONS The network pharmacology analysis reveals the molecular biological mechanism of Artemisia annua anti-NSCLC via multiple active components, multi-channels, and multi-targets. This suggests that Artemisia annua might be developed as a promising anti-NSCLC drug.

A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-nCoV) infected pneumonia (standard version).

In December 2019, a new type viral pneumonia cases occurred in Wuhan, Hubei Province; and then named "2019 novel coronavirus (2019-nCoV)" by the World Health Organization (WHO) on 12 January 2020. For it is a never been experienced respiratory disease before and with infection ability widely and quickly, it attracted the world's attention but without treatment and control manual. For the request from frontline clinicians and public health professionals of 2019-nCoV infected pneumonia management, an evidence-based guideline urgently needs to be developed. Therefore, we drafted this guideline according to the rapid advice guidelines methodology and general rules of WHO guideline development; we also added the first-hand management data of Zhongnan Hospital of Wuhan University. This guideline includes the guideline methodology, epidemiological characteristics, disease screening and population prevention, diagnosis, treatment and control (including traditional Chinese Medicine), nosocomial infection prevention and control, and disease nursing of the 2019-nCoV. Moreover, we also provide a whole process of a successful treatment case of the severe 2019-nCoV infected pneumonia and experience and lessons of hospital rescue for 2019-nCoV infections. This rapid advice guideline is suitable for the first frontline doctors and nurses, managers of hospitals and healthcare sections, community residents, public health persons, relevant researchers, and all person who are interested in the 2019-nCoV.

Gadolinium-chelate functionalized bismuth nanotheranostic agent for in vivo MRI/CT/PAI imaging-guided photothermal cancer therapy.

Multifunctional nanomaterials with simple structure and good biosafety, integrating multimodal imaging and therapeutic functions, can facilitate the development of clinical cancer treatments. Here, a simple but powerful pure bismuth based nanoparticle (Gd-PEG-Bi NPs) was developed from pure Bi NPs and gadolinium-diethylenetriaminepentaacetic acid-bis-tetradecylamide, which not only shows high quality MRI/CT/PAI triple-modal imaging, but can also be a potent photothermal therapy agent under the guidance of the triple-modal imaging. The Gd-PEG-Bi NPs showed good stability and excellent biocompatibility. In vitro and in vivo study demonstrated that Gd-PEG-Bi NPs have ultrahigh X-ray attenuation coefficient, short T1 relaxation time in MRI, and strong PAI signal. Following the imaging diagnosis, the excellent light-to-heat conversion efficiency of Gd-PEG-Bi NPs was capable of suppressing the tumor growth effectively under near-infrared laser radiation in vivo. Such multifunctional nanoparticles were ideal candidates for cancer diagnosis and treatment.

Near-infrared light-triggered theranostics for tumor-specific enhanced multimodal imaging and photothermal therapy.

The major challenge in current clinic contrast agents (CAs) and chemotherapy is the poor tumor selectivity and response. Based on the self-quench property of IR820 at high concentrations, and different contrast effect ability of Gd-DOTA between inner and outer of liposome, we developed "bomb-like" light-triggered CAs (LTCAs) for enhanced CT/MRI/FI multimodal imaging, which can improve the signal-to-noise ratio of tumor tissue specifically. IR820, Iohexol and Gd-chelates were firstly encapsulated into the thermal-sensitive nanocarrier with a high concentration. This will result in protection and fluorescence quenching. Then, the release of CAs was triggered by near-infrared (NIR) light laser irradiation, which will lead to fluorescence and MRI activation and enable imaging of inflammation. In vitro and in vivo experiments demonstrated that LTCAs with 808 nm laser irradiation have shorter T1 relaxation time in MRI and stronger intensity in FI compared to those without irradiation. Additionally, due to the high photothermal conversion efficiency of IR820, the injection of LTCAs was demonstrated to completely inhibit C6 tumor growth in nude mice up to 17 days after NIR laser irradiation. The results indicate that the LTCAs can serve as a promising platform for NIR-activated multimodal imaging and photothermal therapy.

Effects of Weipixiao (胃痞消) on Wnt pathway-associated proteins in gastric mucosal epithelial cells from rats with gastric precancerous lesions.

OBJECTIVE: To study the effects of Weipixiao (胃痞消, WPX) on Wnt pathway-associated proteins in gastric mucosal epithelial cells from rats with gastric precancerous lesions (GPL). METHODS: Sprague Dawley rats were randomly divided into control, model, vitacoenzyme (0.2 g·kg(-1)·day(-1)), WPX high-dose (H-WPX, 15 g·kg(-1)·day(-1)), WPX medium-dose (M-WPX, 7.5 g·kg(-1)·day(-1)) and WPX low-dose (L-WPX, 3.75 g·kg(-1)·day(-1)) groups. After successfully establishing the GPL model, the rats were consecutively administered WPX or vitacoenzyme by gastrogavage for 10 weeks. Differential expression of Leucine-rich repeat-containing G-proteincoupled receptor 5 (Lgr5), matrix metalloproteinase-7 (MMP-7), Wnt1, Wnt3a, and ß-catenin in gastric mucosal epithelial cells in all groups were immunohistochemically detected, and the images were taken and analyzed semiquantitatively by image pro plus 6.0 software. RESULTS: Gastric epithelium in the model group showed significantly higher expression levels of Lgr5, MMP-7, Wnt1, Wnt3a and ß-catenin than those of the control group(P<0.01). Interestingly, we also observed Lgr5+ cells, which generally located at the base of the gastric glandular unit, migrated to the luminal side of gastric epithelium with GPL. The expression levels of Lgr5, MMP-7, Wnt1, and ß-catenin were all down-regulated in the L-WPX group as compared with those of both model and vitacoenzyme groups (P<0.05). A similar, but nonsignificant down-regulation in expression level of Wnt3a was noted in all WPX groups (P>0.05). CONCLUSION: Our findings suggested that the therapeutic mechanisms of WPX in treating GPL might be related with its inhibitory effects on the expressions of Lgr5, MMP-7, Wnt1, ß-catenin and the aberrant activation of Wnt/ß-catenin pathway.

[The effect of baicalin on human brain microvascular endothelial cell under the glucose deprivation combined with hypoxia condition].

OBJECTIVE: To investigate the mechanisms of baicalin on anti-cerebral ischemic through observing the effect of baicalin on human brain microvascular endothelial cell under the glucose deprivation combined with hypoxia condition. METHODS: Human brain microvascular endothelial cells (HBMVECs) cultured in vitro were divided into the following groups: normal group, model group, baicalin high dose group, baicalin middle dose group, baicalin low dose group, nimodipine group. The kits were used to detect the cell viability, leakage rate of lactate dehydrogenase (LDH), Na(+)-K(+)-ATPase, Ca(2+)-ATPase, the concentration of Ca2+ in each group, and apoptosis rates of each group were analyzed by flow cytometry (FCM). RESULTS: Compared with the normal group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase in model group decreased significantly (P < 0.01, P < 0.05). But the leakage rate of LDH, Ca2+ in cells and apoptosis rates increased remarkably (P < 0.01). Compared with the model group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase increased obviously (P < 0.01, P < 0.05) in baicalin high dose group. But the leakage rate of LDH and Ca2+ in cells in baicalin high dose group decreased significantly comparing with that of model group (P < 0.05, P < 0.01). And the reduction of intracellular Ca2+ was superior to that of nimodipine group. Meanwhile, the apoptosis rates decreased significantly in both baicalin high and middle dose groups (P < 0.01). CONCLUSION: Baicalin could improve the cell viability of HBMVECs under the glucose deprivation combined with hypoxia condition. And the mechanisms were related with improving the energy metabolism, inhibiting intracellular calcium overload and decreasing the apoptosis rate of cells further.

[Comparative study on fMRI of the brain based on Dicang (ST 4), Hegu (LI 4) and Houxi (SI 3) on human body].

OBJECTIVE: To observe the correlation relationship between acupuncture at Dicang (ST 4), Hegu (LI 4) and Houxi (SI 3) on the affected side of peripheral facial paralysis patients and activated areas in brain functional areas and central regulation mechanism of acupuncture at Hegu (LI 4) treatment. METHODS: Eighteen cases with left peripheral facial paralysis were randomly divided into a Hegu group, a Dicang group and a Houxi group, 6 cases in each group. They were treated with electroacupuncture at left Dicang (ST 4), Hegu (LI 4) and Houxi (SI 3), respectively, and were examined with fMRI covering the whole brain at the same time. The fMRI data was analyzed by SPM software. RESULTS: It was found that the left precentral gyrus area and the left postcentral gyrus area were activated when electroacupuncture at left Hegu (LI 4), and the right precentral gyrus area and the bilateral postcentral gyrus area were activated when electroacupuncture at left Dicang (ST 4), and there was no activated area at precentral gyrus area and post central gyrus area when electroacupuncture at left Houxi (SI 3). CONCLUSION: The sensory importation information from Hegu (LI 4) and Dicang (ST 4) can converge and coincide in the brain and may influence each other.

Effect of serum from overfatigue rats on JNK/c-Jun/HO-1 pathway in human umbilical vein endothelial cells and the intervening effect of Tongxinluo superfine powder.

OBJECTIVE: To cultivate human umbilical vein endothelial cells (HUVECs) in the serum of overfatigue rats with the intervention of Tongxinluo superfine powder (TXLSP). By examining the variation of the activity of JNK/c-Jun/HO-1 pathway, the possible mechanisms of vascular endothelial dysfunction under overfatigue conditions and the intervening effect of TXLSP were explored. METHODS: The HUVECs were randomly divided into the normal control group, the model group, the SP600125 (a specific antagonist of JNK) group, the TXLSP group and the TXLSP + SP600125 group. The content of carboyhemoglobin (COHb) and the leak rate of lactic dehydrogenase (LDH) in different groups were measured. The mRNA and protein expression of JNK, c-Jun, HO-1 and the phosphorylation level of c-Jun (P-c-Jun) were detected using Western blot and PCR methods. RESULTS: Compared with the normal control group, the COHb level in supernatant was increased significantly in the model group, and the expression of HO-1, JNK, c-Jun mRNA and corresponding proteins and P-c-Jun were also increased remarkably. The increases in these parameters were significantly decreased by SP600125. TXLSP showed remarkable up-regulation on the expression of JNK, c-Jun, P-c-Jun and HO-1 mRNA and their protein expression. Compared with the SP600125 group, the expressions of JNK, c-Jun, P-c-Jun and HO-1 mRNA and its protein in the TXLSP+SP600125 group were significantly increased at different time points (P<0.05, P<0.01). CONCLUSIONS: The vascular endothelial dysfunction under overfatigue conditions is related to the activity of the JNK/c-Jun/HO-1 pathway. One of the mechanisms of TXLSP in improving the vascular endothelial function is to adjust the activity of the JNK/c-Jun/HO-1 pathway at gene and protein levels.

[The endothelium injuries caused by homocysteine and treatmental effects of Tongxinluo powder].

AIM: To observe the effect of homocysteine (HCY) on the function of endothelium cell, and to discuss the possible mechanisms that Tongxinluo super powder affected. METHODS: Healthy male Wistar rats were divided into randomly the control group, the model group, the Tongxinluo group. The effect of Ach on isolated rat thoracic aorta in vitro was examined, the microcirculation was observed by microcirculation meter, the activity of SOD and GSH-PX and content of NO, MDA, ET, Ang II, TXA2, PGI2 was detected. RESULTS: Compared with control group, the effect of Ach on isolated rat thoracic aorta in vitro weakened markablely (P < 0.01), the format and percentage that capillary dilated declined significantly (P < 0.05), after treatment with Tongxinluo powder, the effect of Ach on isolated rat thoracic aorta in vitro was improved obviously (p < 0.01), and the format and percentage that capillary dilated were increased compared with model group; comparing with the control group, the level of Ang II and ET, TXA2 in plasm increased obviously (P < 0.05, P < 0.01), while the content of PGI2 depressed manifestly (P < 0.05), at the same time, both content of NO and activity of SOD, (GSH-PX declined obviously (P < 0.001, P < 0.05). After treatment with Tongxinluo powder, the level of ET, AngII and TXA2 reduced significantly in different degree (P < 0.01), while the content of PGI2 appeared stepping up notably (P < 0.01), and both activity of SOD and NO level increased obviously (P < 0.01, P < 0.05). CONCLUSION: (1) The high homocystein might cause the contracted and dilated function decreased, it might get involved in endothelium disfunction as a result of the massive free radicals production and diastolic-contract factors balance disorder induced by high homocystein. (2) Tongxinluo powder could improve the function of endothelium-dependment dilation induced by high homocystein, that associated with inhibitting the excessive production of free radicals, and improved function of endothelium.

Study on the regulatory effect of electro-acupuncture on hegu point (LI4) in cerebral response with functional magnetic resonance imaging.

OBJECTIVE: To study, through blood oxygen level dependent functional magnetic resonance imaging (BOLD fMRI), the cerebral activated areas evoked by electro-acupuncturing (EA) the right Hegu point (L14) or non-acupoint points on the face, and through comparing their similarities and differences, to speculate on the specific cerebral areas activated by stimulating L14, for exploring the mechanism of its effect in potential clinical application. METHODS: EA was applied at volunteers' right L14 (of 9 subjects in the L14 group) and facial non-acupoint points (of 5 subjects in the control group), and whole brain 3-dimensional T1 anatomical imaging of high resolution 1 x 1 x 1 mm(3) used was performed with clustered stimulatory mode adopted by BOLD fMRI. Pretreatment and statistical t-test were conducted on the data by SPM2 software, then the statistical parameters were superimposed to the 3-dimensional anatomical imaging. RESULTS: Data from 3 testees of the 9 subjects in the L14 group were given up eventually because they were unfit to the demand due to different causes such as movement of patients' location or machinery factors. Statistical analysis showed that signal activation or deactivation was found in multiple cerebral areas in 6 subjects of L14 group and 5 subjects of the control group (P<0.01). In the L14 group, the areas which showed signal activation were: midline nuclear group of thalamus, left supra marginal gyrus, left supra temporal gyrus, right precuneous lobe, bilateral temporal pole, left precentral gyrus and left cerebellum; those which showed signal deactivation were: bilateral hippocampus, parahippocampal gyrus, amygdala body area, rostral side/ audal side of cingulate gyrus, prefrontal lobe and occipital lobe as well as left infratemporal gyrus. In the control group, areas which showed signal activation were: bilateral frontal lobe, postcentral gyrus, Reil's island lobe, primary somato-sensory cortex, cingulate gyrus, superior temporal gyrus, occipital cuneiform gyrus and/or precuneus gyrus and right brainstem; and the area that showed deactivation was left median frontal lobe. CONCLUSION: The effects of EA L14 in regulating cerebral activities could be displayed and recorded through BOLD fMRI, the distribution of signally deactivated area evoked by EA L14 was similar to the known distribution of anatomical orientation of pain in brain, and closely related to the anatomic structure of limbic system, which areas are possibly the acupuncture analgesic effect's cerebral regulating area. Furthermore, activated portion of left central anterior gyrus, which represent the movement of oral facial muscles, and the activated portion of cerebellum are possibly related with the effect of using EA L14 in treating facial palsy and facial muscle spasm. As for the mechanism of signal deactivation of cerebral activities exhibited in the present study that is unable to be elucidated, it awaits for further research.