This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.
Humans ; Female ; Breast Neoplasms/metabolism* ; MCF-7 Cells ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Beclin-1/pharmacology* ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Cell Proliferation

Objective:To establish an ultra-performance liquid chromatography-mass spectrometry （UPLC-MS/MS） for determining the plasma concentrations of 10 active ingredients in Wujiwan at different time points after oral administration， and to compare the pharmacokinetic characteristics between normal rats and rats with chronic visceral hypersensitive irritable bowel syndrome （CVH-IBS）. Method:CVH-IBS rat model was prepared by the neonatal rat colon percutaneous transluminal coronary angioplasty （PTCA） balloon stimulation method. After intragastric administration of Wujiwan （0.245 g·kg^-1）， blood was collected from the jugular vein at different time points， and the plasma concentrations of 10 active ingredients （berberine hydrochloride， palmatine hydrochloride， coptisine hydrochloride， jatrorrhizine hydrochloride， epiberberine， dihydroberberine， evodiamine， evodine， paeoniflorin， albiflorin） in Wujiwan was detected simultaneously by UPLC-MS/MS， the pharmacokinetic parameters of each component in normal rats and CVH-IBS rats were calculated. Result:The established UPLC-MS/MS could sensitively and accurately detect the plasma concentrations of 10 active ingredients of Wujiwan in rats. Compared with the normal group， the absorption rates of these 10 active ingredients of Wujiwan in the blood of CVH-IBS rats all decreased to a certain extent， and the peak time （t_max） was prolonged. Among them， the t_max of berberine hydrochloride and jatrorrhizine hydrochloride were significantly prolonged from 54 minute and 39 minute to 90 minute， respectively （P<0.05， P<0.01）. Area under the plasma concentration-time curve （AUC_0-t） of each component increased， and evodiamine and paeoniflorin were significantly different （P<0.05， P<0.01）. The clearance rates （CL/F） of these 10 active ingredients were all decreased， among which berberine hydrochloride， palmatine hydrochloride and evodiamine had significant differences （P<0.05， P<0.01）. Conclusion:There are significant differences in the pharmacokinetic behavior of the active ingredients in Wujiwan between normal rats and CVH-IBS rats， which may be related to the destruction of microstructure of intestinal epithelial cells and the change of activity of liver enzymes under the pathological state of IBS.

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-1β( IL-1β) 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-1β( 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-1β 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.
Apoptosis ; Kelch-Like ECH-Associated Protein 1/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress ; Plant Extracts ; Signal Transduction ; Tumor Necrosis Factor-alpha/genetics*

The non-specific administration of antitumor drugs is the main cause for the side effects of chemotherapy drugs on normal tissues. The application of nanotechnology in the delivery of anti-tumor drugs is one of the important ways to improve the therapeutic effect and to reduce the side effects. The current study aimed to synthesize pH responsive poly (methoxy-ethylene glycol)-poly(lactic acid)-poly-(β-amino ester) (PBAE) triblock copolymers to deliver docetaxel (DTX) and improve the anti-tumor activity of DTX. PBAE was synthesized by ring opening polymerization and Michael addition reaction, its structure and molecular weight was characterized by ¹H NMR, the dissociation constant of base (pK_b) were determined by acid-base titration method. The critical micelles concentration (CMC) of copolymers was measured by pyrene fluorescence spectroscopy. DTX loaded copolymer micelles were prepared by membrane hydration method. The size and its distribution as well as the stability of micelles were determined by laser light scattering analysis. The drug loading content (DL), entrapment efficiency (EE) and cumulative drug release from micelles were evaluated by high-performance liquid chromatography (HPLC). The sizes of DTX drug-loaded micelles were in the range of 10 to 100 nm with narrow distribution. DL of DTX in PBAE1 and PBAE2 micelles was (5.3 ± 0.10) % and (4.9 ± 0.05) %, respectively, with EE was (93.8 ± 1.70) % and (87.2 ± 4.10) %, respectively. The drug-loaded micelles showed pH sensitive drug release properties under weak acidic conditions, which showed potential drug release of DTX under mild acidic tumor environment. A mouse Lewis lung carcinoma model was established to evaluate the therapeutic efficacy of micellar DTX formulations. Significant inhibitory effect of the nanodrugs was observed with DTX dosages of 10 and 20 mg·kg^-1, respectively. Moreover, the pH responsive PBAE1-DTX micellar drug exhibited stronger therapeutic efficacy on mice xenograft tumor, as compared with the non pH sensitive micellar drug (PELA-DTX) and free DTX. All animal experiments were performed according to the animal ethical standards and approved by the Animal Experiments and Ethical Committee of China Academy of Chinese Medical Sciences (No. 2017090110). The in vivo anti-tumor activity studies showed that the tumor volume growth rates of mice in different drug-administered groups were: PBAE1-DTX 20 mg·kg^-1 < PBAE1-DTX 10 mg·kg^-1 < PELA-DTX 10 mg·kg^-1 < DTX 10 mg·kg^-1 < normal saline, with the PBAE1-DTX group as the most potent group for tumor inhibition. The current pH sensitive DTX nano-micelles showed high potential in further studies to promote the application of nano DTX formulations for tumor treatment.

Docetaxel-loaded nanomicelles were prepared in this study to improve the solubility and tumor targeting effect of docetaxel(DTX),and further evaluate their anticancer effects in vitro. PBAE-DTX nanomicelles were prepared by film-hydration method with amphiphilic block copolymer polyethyleneglycol methoxy-polylactide(PELA) and pH sensitive triblock copolymer polyethyleneglycol methoxy-polylactide-poly-β-aminoester(PBAE) were used respectively to prepare PELA-DTX nanomicelles and PBAE-DTX nanomicelles. The nanomicelles were characterized by physicochemical properties and the activity of mice Lewis lung cancer cells was studied. The results of particle size measurement showed that the blank micelles and drug-loaded micelles had similar particle sizes, ranging from 10 to 100 nm. The particle size of PBAE micelles was changed under weak acidic conditions, with good pH response. The encapsulation efficiency of the above two types of DTX-loaded nanomicelles determined by HPLC was(93.8±1.70)% and(87.2±4.10)%, and the drug loading amount was(5.3±0.10)% and(4.9±0.05)%,respectively. Furthermore,the DTX micelles also showed significant inhibitory effects on Lewis lung cancer cells by MTT assay, and pH-sensitive PBAE-DTX showed better cytotoxicity. The results of flow cytometry indicated that,the apoptosis rate of lung cancer Lewis cells was(20.72±1.47)%,(29.71±2.38)%,and(40.91±1.90)%(P<0.05) at 48 h after treatment in DTX,PELA-DTX,and PBAE-DTX groups. The results showed that different docetaxel preparations could promote the apoptosis of Lewis cells, and PBAE-DTX had stronger apoptotic-promoting effect. The pH-sensitive DTX-loaded micelles are promising candidates in developing stimuli triggered drug delivery systems in acidic tumor micro-environments with improved inhibitory effects of tumor growth on Lewis lung cancer.
Animals ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Docetaxel ; pharmacology ; Drug Carriers ; Lung Neoplasms ; drug therapy ; pathology ; Mice ; Micelles ; Nanoparticles ; Particle Size ; Taxoids

Objective To observe the effects of electroacupuncture on the learning and memory ability and cerebral cortex inflammatory factor of rats with vascular cognitive impairment (VCI); To discuss the mechanism of electroacupuncture for preventing and treating VCI. Methods VCI rat models were made in microemboli injection through internal carotid artery method. The successful modeled rats were randomly divided into model group, positive medicine group and electroacupuncture group, and normal rats were taken as control group. Three days after rat models were established, the positive medicine group was given donepezil hydrochlorideby gavage, and electroacupuncture group was given electroacupuncture at "Baihui" and "Zusanli" acupoints. After treatment, the learning and memory ability was detected by Morris water maze test. The contents of TNF-α, IL-6 and IL-1β in rat brain tissue were detected by ELISA. Results The water maze results showed that with the increase of the number of training, the average escape latency of rats to find platform in positive medicine group and electroacupuncture group all had different degrees of shortening in positioning cruise experiment; in space exploration experiment, positive medicine group and electroacupuncture group to cross the platform area for the first time were significantly reduced compared with the model group; compared with the control group, the contents of TNF-α, IL-6 and IL-1β in the model group were increased significantly; compared with the model group, the contents of TNF-α, IL-6 and IL-β in postive medicine group and electroacupuncture group were decreased. Conclusion Electroacupuncture at "Baihui"and "Zusanli" acupoints can decrease the contents of TNF-α, IL-1 and IL-6 in the cortex of VCI rats, and improve the learning and memory ability of rats.

BACKGROUND:The connection between high-level central nervous system and spinal cord and peripheral nerve under injury level is blocked by traumatic spinal cord injury,and the whole function of the body is thereby influenced.The loss of motor function and feeling both severely affect the patient's life quality in views of physiology,psychology,function and social economics.RNA interference is an effective method to silence target genes,which provides a new treatment strategy for spinal cord injury.OBJECTIVE:To explore the application status of RNA interference in spinal cord injury.METHODS:PubMed and CNKI databases were retrieved using the keywords of "RNA interference,RNAi,spinal cord injury" in English and Chinese,respectively.The articles addressing the application of RNA interference in spinal cord injury were collected and reviewed.RESULTS AND CONCLUSION:Totally 44 articles were enrolled.It is urgent to find a new way to treat spinal cord injury.Compared with the traditional gene silencing technology,RNA interference owing to high specificity,high efficiency,high stability and high penetrability is considered to be a new direction for the studies on the pathogenesis and treatment of spinal cord injury.

BACKGROUNDExcessive iodine intake and viral infection are recognized as both critical factors associated with autoimmune thyroid diseases. Toll-like receptors (TLRs) have been reported to play an important role in autoimmune and inflammatory disorders. In this study, we aimed to clarify the possible mechanism of TLR3 involved in polyinosine-polycytidylic acid (poly(I:C)) promoting excessive iodine intake induced thyroiditis in non-obese diabetic (NOD) mice.

METHODSBoth NOD and BALB/c mice were randomly assigned to four groups: control group (n = 5), high iodine intake (HI) group (n = 7), poly(I:C) group (n = 7) and combination of excessive iodine and poly(I:C) injection (HIP) group (n = 7). After 8 weeks, mice were weighed and blood samples were collected. All the mice were sacrificed before dissection of spleen and thyroid gland. Then, thyroid histology, thyroid secreted hormone, expression of CD3(+) cells and TLR3 as well as inflammatory mRNA level were evaluated.

RESULTSBoth NOD and BALB/c mice from HI and HIP group represented goiter and increasing thyroid relative weight. Thyroid histology evidence indicated that only HIP group of NOD mice showed severe thyroiditis with lymphocytes infiltration in majority of thyroid tissue, severe damage of follicles and general fibrosis. Immunofluorescence staining results displayed a large number of CD3(+) cells in HIP NOD mice. Real-time polymerase chain reaction (PCR) results suggested interferon (IFN)-α increased over 30 folds and IFN-γ expression was doubled compared with control group, but interleukin (IL)-4 remained unchanged in HIP group of NOD mice thyroid. Meanwhile, over one third decrease of blood total thyroxine (TT4) and increased thyroid-stimulating hormone (TSH) was observed in HIP group of NOD mice. Only HIP group of NOD mice represented significantly elevation of TLR3 expression.

CONCLUSIONPoly(I:C) enhanced excessive dietary iodine induced thyroiditis in NOD mice through increasing TLR3 mediated inflammation.

Animals ; Female ; Inflammation ; chemically induced ; metabolism ; Iodine ; toxicity ; Mice ; Mice, Inbred NOD ; Poly I-C ; pharmacology ; Thyroiditis ; chemically induced ; immunology ; metabolism ; Toll-Like Receptor 3 ; metabolism

Background p16INK4a gene plays an important role during the aging and senility.So it was well known to be a leading gene associated with aging.Corneal endothelial cells(CECs) always get trapped in the G1 phase due to the lack of proliferative ability.Whether it is relative with cell senescence is unclear.Objective This study was to investigate the expression ofp16INK4a gene and Bmi1 gene in human CECs from different aged donors ex vivo.Methods The corneal rims,the residual of corneal tissue preserved in DX solution after penetrating keratoplasty,was used in the present study.Parameters were recorded for the donor,including the age,death to preservation interval and preservation to surgery interval.Corneal endothelium survival rate and endothelial cell density were evaluated by trypan blue-alizarin red dying immediately after penetrating keratoplasty.Routine haematoxylin and eosin staining was also performed to proof the normal structure of the cornea.Sections of corneas from different aged donors were classified into ＜30 years group,30-50 years group and ＞50 years group and were immunostained to assess the expressions of p16INK4aprotein,Bmi1 protein and Ki67 protein in CECs.Total RNA was extracted from independent corneal sample for the evaluation of p16INK4a mRNA,Bmi1 mRNA and Ki67 mRNA expression in CECs by quantitative real-time PCR(qRT-PCR).Results The endothelial cell density of each group was (3069 ±172),(2748±64),(2444 ±178)cells/mm2,respectively.Haematoxylin and eosin staining showed the normal structure of corneal epithelium,stroma and endothelium.qRT-PCR examination revealed an age-related increase in p16INK4a mRNA expression in the CECs(F =5.703,P =0.014) and a decrease in Bmi1 mRNA and Ki67 mRNA expression (F =3.950,P =0.042;F=548.500,P =0.000).The further comparison verified a significant elevation in the expression of p16INK4a mRNA in the CECs of the ＞50 years group compared with ＜30 years group and significant decline in Bmil mRNA and Ki67 mRNA the expression (P =0.006,0.013,0.000).Immunohistochemistry in situ confirmed the expression and nuclear localization of p16INK4a protein in CECs,and the expressing intensities of Bmi1 and Ki67 proteins in the elder donors were weaker than those of the younger donors.The immunofluorescence exhibited that the expressing intensity of p16INK4a protein in CECs of 58 years old donor was higher than that of 23 years old donor,showing a consistent result with that of qRT-PCR.Conclusions Expression of p16INK4a gene increases and that of Bmi1 gene decreases upon age.These results suggest that p16INK4a gene is associated with senescence of human CECs.

OBJECTIVETo investigate the etiology, pathology, and clinical characteristics of cryptogenic liver diseases in order to develop a pathogenic profile for clinical diagnosis and therapeutic design.

METHODSThe data of the 566 patients diagnosed with abnormal liver function and who had undergone liver biopsy at our institute between January 2006 to March 2010 were retrospectively analyzed. The Chi-squared (x²) test was used to assess disease correlation with sex and the rank sum test was used to assess disease correlation with continuous data since all data had asymmetric distribution.

RESULTSAmong the 566 patients, abnormal liver function was attributed to alcoholic liver disease (n=175; 30.92%), drug-induced or environmentally-induced liver disease (n=101; 17.84%), hereditary and metabolic disease (n=93; 16.43%), infectious hepatitis disease (n=84; 14.84%), fatty liver disease (n=53; 9.36%), and autoimmune liver disease (n=30; 53.00%). Thirty patients had unknown etiology, despite liver biopsy analysis. Among these disease subgroups, there were distinct correlations with sex, age, and levels of alanine transaminase (ALT) and gamma-glutamyltransferase (GGT). The autoimmune liver disease group was correlated with sex (q=9.14, 7.435, 5.071, 9.529, and 12.5, respectively; P less than or equal to 0.01). The alcoholic liver disease group and autoimmune liver disease group were correlated with age (vs. genetic metabolic disease group: q=17.254 and 10.302; infectious hepatitis group: q=17.523 and 10.697); drug/environmentally-induced liver damage group: q=9.170 and 5.266); fatty liver group: q=7.118 and 4.661) (P less than or equal to 0.01). In addition, the alcoholic and autoimmune liver disease groups were correlated with GGT levels (vs. genetic metabolic disease group: q=8.003; infectious hepatitis group: q=4.793; drug/environmentally-induced liver damage group: q=4.404) (P less than or equal to 0.01).

CONCLUSIONLiver pathology is important for the diagnosis of cryptogenic liver diseases. Patient age, sex, and biochemistry index may facilitate diagnosis and treatment in the absence of pathology.

Adolescent ; Adult ; Biopsy ; Child ; Child, Preschool ; Female ; Humans ; Liver ; pathology ; Liver Diseases ; classification ; diagnosis ; pathology ; Male ; Middle Aged ; Young Adult