Objective To understand the molecular mechanism by which protein kinase C? mediates multidrug resistance of human glioma cell line SHG-44.Methods SHG-44/ADM was constructed by stepwise concentration increasing method and intermittent administration method.SHG-44/WT and SHG-44/ADM were treated by PKC? reactivator PMA and PKC? inhibitor staurosporine,then the expressions of PKC? and MDR-1 were detected by Western blotting,the PKC? activity was assayed by kinase,and ADM accumulation was determined by fluorescence spectrometry.Results PMA increased PKC? activity and MDR-1 protein expression and activity.Staurosporine was able to block PKC? activity and decrease MDR-1 expression and activity.Conclusion Multidrug resistance in human glioma cells is mediated by PKC? via MDR-1 pathway.

[ ABSTRACT] AIM:To observe the inhibitory effect of madecassoside on the LPS-stimulated microglia and to inves-tigate its possible mechanism.METHODS:Microglia cells of neonatal Sprague-Dawley ( SD) rats were cultured, isolated and purified.Microglia cells were activated with lipopolysaccharide ( LPS) .The inhibitory effect of madecassoside on micro-glia was measured by MTT assay.Tumor necrosis factor alpha (TNF-α), interleukin 1β(IL-1β) were detected by ELISA. Cell cycle and apoptotic rate were evaluated by flow cytometry.The expression of TLR4 was detected by Western blotting. The expression of NF-κB was detected by RT-PCR.RESULTS: LPS induced the proliferation of microglia and release in-flammatory cytokines significantly.Compared with LPS group, madecassoside inhibited the proliferation of microglia induced by LPS in a dose dependent manner.The IC50 value of madecassoside was 10.97 nmol/L to microglia after incubation for 48 h.Madecassoside also decreased the levels of TNF-αand IL-6, increased the ratios of microglia at the G2 phase and the ap-optotic rate, decreased the expression of TLR4 and NF-κB significantly (P<0.05).CONCLUSION:Madecassoside has in-hibitory effects on the proliferation of LPS-stimulated microglia, by which the mechanism may be related to inhibition of the expression of TLR4 and NF-κB, change of cell cycle distribution and induction of microglia apoptosis.

An auxiliary training system was explored for the development of creativity of long-termsystem medical undergraduates through scientific researches in spare time.In practice,professionals from research groups of different disciplines were recruited for developing wide-scope theoretical and technical bases of the undergraduates through training among these research groups followed by focused researches on topics of the running projects of these professionals.The undergraduates were engaged in a serial of research activities,including the discovery of problems for reasoning out scientific research projects,the writing of their project proposals,the summary of their data and the writing of reports,to develop their creativity and their qualification for scientific researches.

BACKGROUND:Cervical cancer is one of the most frequent malignancies in women worldwide, and its occurrence and development is closely related to cyclooxygenase-2 (COX-2).OBJECTIVE: To examine the expression of COX-2 alternative splicing variants in human cervical carcinoma tissue and understand its possible implications.DESIGN: Non-randomized controlled experiment.SETTING: Key Laboratory of Biochemistry and Molecular Pharmacology,Department of Obstetrics and Gynecology, First Affiliated Hospital,Chongqing Medical University.PARTICIPANTS: Carcinoma tissue and normal tissue were obtained from 13 cervical carcinoma patients admitted during March 2002 to April 2002in the Department of Obstetrics and Gynecology, First Affiliated Hospital,Chongqing Medical University.METHODS: A pair of specific primers were designed for reverse transcription-polymerase chain reaction (RT-PCR) to obtain the mRNA of COX-2 in human cervical carcinoma tissues. The resultant band on electrophoresis was cloned, sequenced and analyzed.MAIN OUTCOME MEASURES: ① Agarose gel electrophoresis result of the PCR product of carcinoma and normal tissues; ② Sequencing result of the electrophoresis band from carcinoma and normal tissues.RESULTS: No COX-2 band (252 bp) was found in electrophoresis for normal tissues, while 2 bands appeared for cervical carcinoma tissues, including a new electrophoresis band of 534bp besides the COX-2 band. Cloning and sequencing revealed that this new band contained not only exons 7and 8 of COX-2 gene but also a reserved intron of 282 bp intron between exons 7 and 8. Analysis of the predicted amino acid sequence indicated that an in-frame stop codon occurred in the 48-50 bp of the intron retained in the mRNA.CONCLUSION: The presence of COX-2 alternative splicing mRNA variant (Genbank accession number:BU493602)is confirmed in human cervical carcinoma tissue, which codes for a protein possibly smaller than COX-2.

AIM: To observe the anti-inflammatory effect of CQMUH-011 and to explore its mechanism.METHODS: Three kinds of animal models, mouse ear swelling induced by xylene, rat granuloma induced by cotton ball and rat rheumatoid arthritis induced by Freund's complete adjuvant, were established to study the anti-inflammatory effect of CQMUH-011.The ear swelling degree, dry weight of cotton ball granuloma, arthritis index, paw swelling and ankle joint pathological changes were measured to reflect the severity of inflammation.The anti-inflammatory mechanisms of CQMUH-011 were investigated by detecting the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by ELISA.Malondialdehyde (MDA), myeloperoxidase (MPO) and nitric oxide (NO) were determined by corresponding kits.RESULTS: Treatment with CQMUH-011 significantly decreased TNF-α, IL-6 and NO concentrations, MDA contents and MPO activity in the serum.Meanwhile, Ear swelling degree, dry weight of cotton ball granuloma, arthritis index, paw swelling and ankle joint pathological damage were attenuated.CONCLUSION: CQMUH-011 has an anti-inflammatory effect, which may be related to inhibiting the production of inflammatory factors and attenuating lipid peroxidation.

To explore the role of autophagy in hair follicle cycle and whether 3-methyladenine(3-MA)could promote hair regeneration in C57BL/6 mice through inhibiting autophagy flux, hair regeneration model of C57BL/6 mice was induced on the dorsal skin by depilation, and 3-MA was intraperitoneally injected to investigate hair regrowth, meanwhile vehicle and rapamycin(RAPA)were used as the controls. Results showed that 3-MA could obviously promote hair regrowth in depilated C57BL/6 mice. Furtherly, haematoxylin-eosin staining, immunohistochemical staining, immunofluorescence and Western blot tests were used to investigate the autophagy signals and the cell proliferation. Results showed that the expression of Beclin1 and LC3B II/I ratio were significantly decreased. Expression of P62 and Ki67 were increased, as well as the CD34 and CD49f double-labeled hair follicle stem cells were obviously increased inside bulge areas in 3-MA group, while contrarily in RAPA group. These results affirmed 3-MA, an autophagy inhibitor, could promote hair regeneration in depilated C57BL/6 mice by facilitating the transformation of hair follicle from telogen to anagen. 3-MA and other analogous autophagy inhibitors probably have a potential usage in future therapy in human telogen effluvium diseases.

To explore the pharmacological effect of 3,4-dihydroxyacetophenone (DHAP) on the apoptosis of RAW264. 7 macrophage cells and the mechanism, RAW264. 7 macrophage cells were treated with 100 or 500 mg/L lipopolysaccharide (LPS), with or without 10-5 mol/L DHAP for 24h. Trypan blue dye exclusion assay was used to assess cell viability. Cell apoptosis was morphological studied and flow cytometric assay was used. Tumor necrosis factor-α (TNF-α) level was measured by ELISA methods. IκB protein was determined by Western blotting. Our results showed that in 100 mg/L LPS-stimulated macrophages, DHAP enhanced the cell apoptosis while in 500 mg/L LPS-stimulated macrophages, DHAP significantly inhibited the cell apoptosis. In both groups,DHAP increased the level of IκB but decreased the level of TNF-α. It is concluded that DHAP has dual effect on the apoptosis of RAW 264.7 cells treated with different concentrations of LPS. This effect may be due to the inhibition of activation of NF-κB and autocrine production of TNFα. Our study suggests that DHAP may have anti-inflammatory effect on LPS-activated macrophages.

AIM: To investigate the effect of epinephrine on LPS-induced pro-inflammatory mediators (TNF-?, NO and COX-2) and anti-inflammatory mediators (HO-1 and IL-10) production in murine macrophage RAW264.7 cells, and to determine whether these effect is due to the influence of epinephrine on NF-?B activation. METHODS: RAW264.7 cells were cultured in vitro with 10 ?g/L LPS in the absence or presence of epinephrine at variant concentrations (1, 5, 10, 50 ?mol/L) for 24 hours, then the supernatants was collected for measuring TNF-? and IL-10 by ELISA and Griess reagent was used to measure NO (NO_2-/NO_3-) concentration. At the same time point, cells were harvested and COX-2, HO-1 and I?B-? was detected by Western blotting. RESULTS: 10 ?g/L LPS significantly induced the production of TNF-?, NO (NO_2-/NO_3-), COX-2, HO-1 and IL-10. When epinephrine was added into the medium together with LPS, the pro-inflammatory mediators production was decreased in a dose-dependent manner, however, anti-inflammatory mediators HO-1 and IL-10 expression was enhanced by epinephrine. Epinephrine has no significant effect on I?B-? degradation in LPS-activated RAW264.7 cells. CONCLUSION: Epinephrine down-regulates LPS-induced pro-inflammatory mediator expression while promotes anti-inflammatory mediator production in murine macrophages. These effect seems to be independent of NF-?B activation.

In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.
Amino Acid Sequence ; Base Sequence ; Cyclooxygenase 2/*biosynthesis ; Cyclooxygenase 2/genetics ; Labor Onset/*metabolism ; Molecular Sequence Data ; Myometrium/*enzymology ; Myometrium/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Sequence Analysis

OBJECTIVEThe present study is to investigate the protective effects of asiaticoside on sepsis-induced acute kidney injury in mice.

METHODWith the sepsis induced by cecal ligation and puncture (CLP), forty eight kunming mice were randomly divided into four groups as sham operated, CLP treated, CLP + asiaticoside 15, 45 mg x kg(-1) groups. General conditions and the amount of dead rate of mice were observed. The BUN and Cr levels were observed by the kits. IL-6 in serum was assayed by enzyme-linked immunosorbent assay (ELISA). Kidney tissues were harvested for determination of iNOS expression by Western blotting analysis. The pathologic changes were observed under electron microscope via hematoxylin-eosin (HE) stain.

RESULTCompared with CLP group, the death rate, the levels of BUN, Cr, IL-6, and iNOS protein expression of asiaticoside groups were significantly reduced. The pathologic changes in kidney tissues induced by sepsis were significantly attenuated dose-dependently by asiaticoside under electron microscope.

CONCLUSIONAsiaticoside has protective effects against sepsis-induced acute kidney injury, which were probably associated with the inhibition of IL-6 in serum and iNOS protein in kidney tissues.

Acute Kidney Injury ; drug therapy ; etiology ; prevention & control ; Animals ; Disease Models, Animal ; Female ; Humans ; Male ; Mice ; Plant Extracts ; administration & dosage ; Random Allocation ; Sepsis ; complications ; Triterpenes ; administration & dosage