The Effect Of EGb761 On The Expression Of HMGB1 In LPS-induced THP-1

Studybackround:The sepsis is a leading cause of death in non-coronary intensive care units (ICU). The overall mortality rate at 30%-40%, the mortality rae more than 70% in older patients or associated with underlying health problems of patients.According to a new epidemiological survey, In the United States, the incidence of severe sepsis is estimated to be 300 cases per 100000 population. Approximately half of these cases occur outside the ICU. A fourth of patients who develop severe sepsis will die during their hospitalization. Septic shock is associated with the highest mortality, approaching 50%.In the pathogenesis of sepsis,both domestic and foreign scholars have been carrying out large-scale research in clinicaling trials and basicing experiment,however, no reached a consensus conclusion. The tradition says sepsis is an important cause of organ injury of patients with excessive response of inflammatory cells and the inflammatory mediators. However recent domestic and foreign scholars have found that HMGB1 protein is late medium for sepsis, which rises in the majority of patients with sepsis. Synthesis of HMGB1 by immune cell activation, may be associated with TLR-2, TLR-4 occurs, thus initiating the inflammatory reaction similar to LPS. HMGB1 will also cause the coagulation factor and neutrophil aggregation, compared with the proinflammatory cytokines IL-1, LPS stimulated the release of HMGB1 occurred at a relatively late stage. Although HMGB1 increased in patients with severe sepsis, but on the sepsis prognosis and no prognostic significance.The experimental mice had dead after injected with HMGB1,however the HMGB1 antibody has certain therapeutic effecting that LPS and polymicrobial sepsis induced by cecal ligation and puncture of mice, therefore the concluded that HMGB1 may be a new target for the treatment of sepsis.The Herbal extract of Ginkgo biloba (extract of Ginkgobiloba, EGb761) is a natural substance extracted from the leaves of Ginkgo biloba, which is the main effective components flavonoids glycosides (24%) and cyclohexene in mushroom vinegar (6%), EGb761 is a kind of pure natural antioxidants, regulate a variety of antioxidant enzyme activity, anti-inflammatory, anti-cancer, anti-aging inhibition of platelet aggregation, pharmacological activities.Ginkgo biloba(EGb761) has been used in traditional Chinese medicine for thousand years. EGb contains 24% flvonol glycosides (the flvonoid fraction) and6% terpene lactones (terpenoid fraction).Its purported biological effcts include freer adical scavenging,antiapoptotic, anti-inflmmatory, inhibit platelet aggregation, and present other pharmacological effects antioxidative activities. One study has already shown that EGb761 could inhibit the expression levels of IL-1β、IL-6、TNF-α,but the international scholar shows that EGb761 can reduce the acute lung injury induced by LPS, However, up to now there still lack of relatively systematic reports about the effect of EGb761 on the expression of HMGB1 in LPS-induced THP-1 at the country.Objective:through the experiment, THP-1 cells by culture in vitro, adjust the observation of Ginkgo biloba extract EGb761 on LPS induced release of HMGB1 protein expression in THP-1 cells.For the further experiment and later clinical application and provide a feasible basis.Materials and methods:1 experimental cell and grouping1.1 THP-1 cell cultureTo generate cells were seeded in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum (FCS) in a CO2(5%) incubator at 37℃.Seed 1.0-1.5×107 cells into a 75 cm2 tissue culture flask or 2.5×107 cells into a 150-cm2 tissue culture flask in RPMI-1640 medium and add 10%(v/v) FCS for 48 hr in a 5% CO2atmosphere.. according to the cell growth and changed of liquid for repeating it two or three times a week.1.2 The THP-1 were randomly divided into 5 groups (n=3)(1)The inflammatory factor detection of group:THP-1+EGb761(50μg/ml)+LPS(1 μg/ml)(2)Activation of NF-kappa B testing group:THP-1+EGb761(50μg/ml)+LPS(1μg/ml)(3)At different time points HMGBlprotein detection group:THP-1+LPS(1μg/ml)(4) Of different concentration of EGb761 on the testing group HMGB1:THP-1+EGb761 (50μmg/ml,100μg/ml,150μg/ml)and LPS(1μg/ml) (5)Nuclear translocation of HMGB1:THP-1+EGb761group(50μg/ml)+LPS(1μg/ml)2 Observation index2.1 The expression levels of IL-1β、IL-6、TNF-α proteinA proper dose of THP-1 cell was inoculated into 24 hole cell culture plate and different concentrations of EGb761 were added for pre-processed 2h,then the THP-1 cells were induced by LPS(1μg/ml) for a certain period of time(6h、12h). to generate the liquid of cells that the amount of TNF-α, IL-1β, and IL-8 in culture supernatants was determined using the ELISA kit according to the manufacturer’s protocol.2.2 The expression levels of NF-κB in different time points by induced THP-1 cellsA proper dose of THP-1 cell was inoculated into 24 hole cell culture plate and different concentrations of EGb761 were added for pre-processed 2h,then the THP-1 cells were induced by LPS(1μg/ml) for a certain period of time(4h、6h、12h). To collect the nuclear and cytoplasmic extracts, cells were harvested using the NE-PER Cytoplasmic and Nuclear Protein extraction kit according to the manufacturer’s instructions. Protein concentrations of the supernatant were determined with a Bradford Protein Assay Reagent kit.transferred onto polyvinylidene difluoride membranes in a wet-transfer apparatus. Membranes were blocked with 5% nonfat milk in TBS-Tween (TBS-T) for 1 h and then incubated with a specific primary antibody overnight at 4℃. Membranes were washed three times with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h, and blots were developed with enhanced chemiluminescence reagents and exposed to x-ray film. Relative intensities were measured to quantify protein levels using Image-Pro Plus software. All blots were quantified and normalized against an internal control to adjust for the amount of proteins loaded.2.3 The expression levels of HMGB1 in different time points by Western blottingA proper dose of THP-1 cell was inoculated into 24 hole cell culture plate and different concentrations of EGb761 were added for pre-processed 2h,then the THP-1 cells were induced by LPS(1μg/ml) for a certain period of time(6h、12h、18h). To collect the nuclear and cytoplasmic extracts, cells were harvested using the NE-PER Cytoplasmic and Nuclear Protein extraction kit according to the manufacturer’s instructions. Protein concentrations of the supernatant were determined with a Bradford Protein Assay Reagent kit.transferred onto polyvinylidene difluoride membranes in a wet-transfer apparatus. Membranes were blocked with 5% nonfat milk in TBS-Tween (TBS-T) for 1 h and then incubated with a specific primary antibody overnight at 4℃. Membranes were washed three times with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h, and blots were developed with enhanced chemiluminescence reagents and exposed to x-ray film. Relative intensities were measured to quantify protein levels using Image-Pro Plus software. All blots were quantified and normalized against an internal control to adjust for the amount of proteins loaded.2.4 The effect of different concentrations of EGb761 on the expression levels of HMGB1 in LPS-induced THP-1 by Western blottingA proper dose of THP-1 cell was inoculated into 24 hole cell culture plate and different concentrations of EGb761(10μg/ml、100μg/ml、150μg/ml)were added for pre-processed 2h,then the THP-1 cells were induced by LPS(1μg/ml) for a certain period of time(18h). To collect the nuclear and cytoplasmic extracts, cells were harvested using the NE-PER Cytoplasmic and Nuclear Protein extraction kit according to the manufacturer’s instructions. Protein concentrations of the supernatant were determined with a Bradford Protein Assay Reagent kit.transferred onto polyvinylidene difluoride membranes in a wet-transfer apparatus. Membranes were blocked with 5% nonfat milk in TBS-Tween (TBS-T) for 1 h and then incubated with a specific primary antibody overnight at 4℃. Membranes were washed three times with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h, and blots were developed with enhanced chemiluminescence reagents and exposed to x-ray film. Relative intensities were measured to quantify protein levels using Image-Pro Plus software. All blots were quantified and normalized against an internal control to adjust for the amount of proteins loaded.2.5 The nuclear translocation of HMGB1 in the THP-1 cells induced by LPSA proper dose of THP-1 cell was inoculated into 24 hole cell culture plate and different concentrations of EGb761(150μg/ml)were added for pre-processed 2h,then the THP-1 cells were induced by LPS(1μg/ml) for a certain period of time(18h).using the confocal microscopy fluorescence marker, HMGB1 protein with green fluorescent labeling, nucleus blue fluorescent labeled as control, the two overlap observed under a fluorescence microscope afterdistribution of HMGB1 in cells.3. statistical analysisMean standard deviation of each date using(mean+SD)representation and application SPSS 13.0 statistical sofeware for processing. Analysis and comparison between different groups by single factor variance,with(P<0.05)for the difference had statistical significance.4. Result4.1. The expression levels of IL-1β、IL-6、TNF-α protein by ELISAELISA results showed that THP-1 cells induced by LPS 6-12h,which produce large amounts of IL-1,IL-6,TNF-α inflammatory factor higher than contrast group (P<0.05).Training significantly increased IL-1,IL-6,TNF-α,concentration in the solution, through the intervention of EGB761, THP-1 cell culture, IL-6,IL-1,TNF-concentration solution were significantly lower than the LPS group have significant difference (P<0.05).4.2. The expression levels of NF-κB in different time points by induced THP-1 cellsWestern blotting detection of THP-1 cells induced by LPS, increased expression of NF-κB,after EGB761 intervention in 2-12h, THP-1 cell culture medium NF-kappa B content with EGb761 intervention prolonged compared with blank control group significantly decreased(P<0.05) had statistical significance.4.3. The expression levels of HMGB1 in different time points by Western blottingWestern blotting testing results showed that the LPS induced THP-1 cells afterl2h,compared with the blank group increased HMGB1 protein content in the supernatant was, and with the prolongation of time 18hHMGB1 protein content significantly increased (P<0.05)with statistical significance.4.4. The effect of different concentrations of EGb761 on the expression levels of HMGB1 in LPS-induced THP-1 by Western blottingThe HMGB protein expression showed that EGb761 intervention in LPS induced THP-1 cells after 18h of HMGB1 in the supernatants of low protein content compared with blank control group, HMGB1 protein content as the EGB761 concentration increased to 150μg/ml were decreased in a dose-dependent effect (P<0.05) there was statistical significance.4.5.EGb761 inhibited LPS induced THP-1 cells to release nuclear translocation of HMGB1 proteinThe confocal microscope display that blank group HMGB1 distribution is almost all in the nucleus; LPS induced group showed most of HMGB1 from the nucleus to the cytoplasm in the transfer; EGb761 and LPS intervention group were visibled part of the HMGB1 distribution.Conclusions:1.LPS may induce the expression of IL-1β,IL-6, TNF-α and the activation of NF-κB in the LPS-induced THP-1 cells to promote the expression and nuclear translocation of HMGB 1.2. EGb761 may inhibit the progress of expression and activation mentioned above, thus affect the expression and the progress of nuclear translocation of HMGB 1.