| BackgroundListeria monocytogenes (LM) is the only pathogenic type of Listeria to human beings. In clinical treatment, it is still a difficult challenge for us to overcome the high morbidity and mortality of LM infections, even though antibiotics have been widely used in treatments for infectious diseases. LM infections mostly strike populations with low immunity, such as the aged, children, expectant mothers, people who take immunosuppressants or with immuse syetem diseases. In general, LM infections are caused by digestive tract infections. In detail, LM can adhere to and invade intestinal epithelial cells once people take LM polluted food. In this process, Internalin B (InlB), which exists on the surface of LM, is the key toxicological facter. By imilating Hepatocyte growth factor or Scatter factor, it can unite with the Hepatocyte growth factor receptor or Scatter factor receptor (c-Met) which exist on intestinal epithelia. Then, InlB will trigger continuous signaling cascades in the same way of the natural ligands as HGF/SF, and induce conversions from tyrosine residues to phosphoric acid on c-Met β chains, including the activation of c-Met, collection of Gabl, Cbl and Shc, phosphorylation, and the formation of p85 complexus which contains adaptors and PI3K sigmasubunits. Thereupon, LM will invade into the cells and cause further infections. Unfortunately, intestinal epithelial cells lack phagocytic ability, so LM can achieve long term survival and perliferation after the invation. Further more, LM can trigger cross infections between the cells, and get to tissues and organs by entering blood, which ends into multiple infections. Among them, meningitis, blooding poisoning, abortion and focal infections are the most common kinds of listeriosis, and meningitis is the most serious one which has the highest fatality rate. Its occurrence bases necessarily on the condition that human brain microvascular endothelial cells (HBMECs) being invaded by LM, because brain microvascular endothelial cells (BMECs) are the most important components of blood-brain barrier (BBB). During which, the key step is that LMs have to reach the brain through blood circulation and pass through the BBB. In a word, the LM caused meningitis bases on the following indispensable conditions. Firstly, LMs adhere to, invade and proliferate in intestinal epithelia after LM polluted food being esten by people with low immunity. Secondly, LMs enter blood by passing trought intestinal epithelial cells, and cause serious blood poisoning as well as multiple infections of tissues and organs. Finally, LMs invade and proliferate in BMECs, then pass throught BBB and result in meningitis.LM is a kind of intracelltdar bacterium which is devided into obligate intracelltdar bacterium and facultative intracelltdar bacterium. Among them, an obligate intracelltdar bacterium can only parasitize in living cells, while a facultative intracelltdar bacterium can parasitize in both living cells and extracellular abiotic conditions. LM is just a kind of facultative intracelltdar bacterium, it can be cultured in extracellular abiotic media with relatively low requirements for nutrition and temperature. In general, LM is proliferative even in a condition of 4℃. Thus, it is of relatively great viability which results in high contamination rate for food and high infection rate for people.At present, even though antibiotics have been widely and efficiently used in treatments for infectious diseases, as for those caused by Listeria, the applicable antibiotics are only extracellular ones which are incapable of entering the cells, such as penicillins, cephalosporins, aulfamethoxazole etc. Thus, LMs will not be killed by the antibiotics once they enter the cells. In addition, it is known that intracelltdar bacteria can escape from body immunity and elimination. As a result, LM causes relatively high morbility and mortality to the infected people. To face the severe challenge of a lack of effective medicines for LM infections, we propose a treatment method which makes use of c-Met receptor blockers to interrupt the uniting of InlB and c-Met. This method is based on the mechanism that InlB can imilate Hepatocyte growth factor to unite with Hepatocyte growth factor receptor or Scatter factor receptor/c-Met, thus realizing invasion of the cells and activation of the signal paths. The effects of c-Met receptor blockers in restraining LM from invading the cells and in treating the corresponding infections are studied in this research.As we know, geldanamycin is a kind of classic medicine of the c-met receptor inhibitors, but it has low water solubility and the drawbacks of higher toxicity on hepatic and renal which lead to rarely used currently in clinical treatment. However, geldanamycin analogues (GAA), such as 17-allylaminogeldanamycin (17-AAG), 17-dimethy-laminoethylamino-17-demethoxygeldanamycin (17-DMAG), have higher water solubleness, lower hepatotoxicity and renal toxicity, etc, so they can be used as potential c-Met inhibitors for research and development in blocking LM adhering to receptors on the surface of cells. At the same time, we know about a new type of c-Met receptor blocking agent cabozantinib (XL-184), which is approved by FDA in 2012 for clinical treatments of diseases. XL-184 has longer biological half-life and lower side effects than the GA. Facing the dilemma of no effect methods against severe LM infection, XL-184 can be a potential new antibacterial drug to be further studied on prevention and treatment of LM infection.Consequently, according to the clues above, this paper focus on the following aspects:(1) by culturing Caco-2 cells and human brain microvascular endothelial cells (HBMECs) in vitro, the effects of 17-AAG and XL-184 in restraining LM from invading the cells are studied; (2) By inoculating Caco-2 cells and HBMECs in Transwell chambers, and measuring the unilaminar transepithelial electrical resistance (TEER) values and osmoses of 17-AAG and XL-184, effects of the c-Met receptor blockers on integrity protection of the cell barriers are studied; (3) by culturing Caco-2 cells and HBMECs in vitro, and using fluorescence staining and lactic dehydrogenase (LDH) release level detection, the cell mortality rates are studied in different medications of 17-AAG and XL-184.Methods1. Researches on the relationship between LM concentration and optical density (OD)First,100 μl LM bacterial liquid is put into a centrifugal machine when the cultured LMs reach logarithmic phase. The centrifugal speed in set to 10000 rpm, the supernatant is then removed by centrifugation. Second, the bacterial liquid is diluted to 1×101～10, then, ODs of the diluted liquids are meatured by using an ultraviolet spectrophotometer at 600 nm. Next,100 μl bacterial liquids with different concentrations are smeared on antibiotics-less BHI solid media respectively, total bactrical counts are then recorded after the media being put in an incubator for 24 h. Finally, relationships between the total bactrical counts, the bacterial liquid dilutabilities and the ODs are analysed and drawn by standard curves.2. Researches on the effects of c-Met receptor blockers on growth curves of LMFirst,5 ml BHI liquid media with different XL-184 concentrations (i.e.0,0.05, 0.1,0.25,0.5,1,2.5,5,10 μM) are prepared. Meanwhile,100 μl LM bacterial liquid in logarithmic phase is added into each medium. After that, the LMs are incubated in a shaker under a condition of 37℃ and 200 rpm. Then,100 μl bacterial liquids with different lengths of incubation time (i.e.0,2,4,6,8,10,12,14,16h) are put in a 96-well plate, followed by meaturing their absorbance values by using a microplate reader at 600 nm. Finally, the growth curves of LM are drawn by taking time as x-coordinate, while the absorbance values as y-coordinate.3. Researches on the effects of LM in invading Caco-2 cells and HBMECsFirst, Caco-2 cells and HBMECs are cultured respectively in 24-well plates until they form compact monolayers (about 105 per well). Second, antibiotics-less media and LMs (about 107 per well, ensuring a multiplicity of infection of 100) are added to the monolayer cells, they are then cultured under a condition of 37℃ and 5% CO2 for 90 min. Next, the cells are light washed with sterile PBS three times, and incubated for 1 h after adding in media with 100 μg/ml gentamicin. This step can restrain extracellular bacteria. After that, the cells are light washed with sterile PBS three times again, followed by adding in 0.5%Triton X-100(100 μl per well).8 min later,50 μl distilled water are added in to end the craking function of Triton X-100. Finally, the cell lysates are diluted gradiently (101～104), and the LM invasion rates are then counted after the lysates being cultured on coated plates for 24 h.4. Researches on the effects of c-Met receptor blockers in restraining LM from invading Caco-2 cells and HBMECs in vitroFirst, Caco-2 cells and HBMECs are cultured respectively in 24-well plates until they form compact monolayers (about 105 per well). Second, the cells are incubated under different conditions. In details, different incubation time, different concentrations of 17-AAG or XL-184 alone or in combination with Ampicillin (AMP) being added in. After that, LMs (about 107 per well, ensuring a multiplicity of infection of 100) are added in. The cells are then light washed with sterile PBS three times, followed by adding in media with 100 μg/ml gentamicin.1 h later, the cells are craked by using 0.05%Triton X-100 after being light washed three times. Finally, the cell lysates are diluted gradiently(101-104), and the LM invasion rates are then counted after the lysates being cultured on coated plates for 24 h.5. Researches on the effects of c-Met receptor blockers on permeability of monolayer cellsFirst, Caco-2 cells and HBMECs are inoculated on the upper layers of Transwell chambers respectively. The cells would form compact monolayer barriers which can prevent small molecular substances from passing through. Meanwhile, the barriers separate the Transwell chambers into upper chambers and lower chambers. Thereupon, blood-intestine barriers and blood-brain barriers are simulated by the Caco-2 cell barriers and HBMEC barriers respectively. Second, MEM media with different concentrations of XL-184 and 1640 media with different concentrations of 17-AAG are added into the upper chambers. After being incubated for 24 h, the cells are light washed with sterile PBS three times, bacteria (MOI=100) are then added in. 1.5 h later,3μg HRP is added in, and the cells are then incubated at 37℃. Next,30μl media are taken from the lower chambers after different time (i.e.0,2,4,6 h), then, they are cultured on antibiotics-less BHI solid plates at 37℃ for 24 h. Finally, total bactrical counts on the plates are recorded, HRP concentrations of the MEM media and 1640 media in the lower chambers are measured by using a microplate reader at 405 nm, and TEER values of the cells are measured by using a resistance meter to reflect changes of permeability of the monolayer cells. It is noteworthy that the media in the lower chambers should be kept at 2 ml by adding in sterile media.6. Researches on the effects of c-Met receptor blockers on LDH release rateFirst, MEM media with different XL-184 concentrations and 1640 media with different 17-AAG concentrations are used to culture Caco-2 cells and HBMECs, and two different culture time are adopted (i.e.1 h and 10 h). After that, the cells are light washed with sterile PBS three times, followed by adding in 1×107 LM (MOI=100). Then, the cells are cultured at 37℃ for 1.5 h. Next,10 ul supernatants of the cells are put in 96-well plates to measure its LDH concentration. Afterwards,10% TritonX-100 are added in to crake the cells, the lysates are then put in 96-well plates to measure the intracellular LDH concentration. The measurement principle, steps and methods are presented in the reagent’s introduction, the absorbance values are measured at 450 nm.7. Analyses of cell viabilityFirst, MEM media with different XL-184 concentrations (i.e.2.5 μM,0.5 μM and 0 μM) and 1640 media with different 17-AAG concentrations (i.e.1000,250 and 0 nM) are used to culture Caco-2 cells and HBMECs, and two different culture time are adopted (i.e.1 h and 10 h). After that, the cells are light washed with sterile PBS three times, followed by adding in 1×107 LM (MOI=100). Then, the cells are cultured at 37℃ for 1.5 h. Next, they are light washed with sterile PBS three times again, followed by adding in 15 μl diluted DAPI dye liquor and 15 μl diluted PI dye liquor in a dark chamber.30 min later, cell viability is measured by using an OLYMPUS laser-scanning confocal microscopy.8. Statistical analysisSPSS 13.0 is usd for statistical analysis. The measurement data are formatted as x±s, where x denotes mean values, and s denotes standard deviations. One-way ANOVA and two-way ANOVA are used. P<0.05 is considered statistically significant. All the experiments are repeated at least three times.Results1. C-Met inhibitor have no effects on the growth curves of LM in vitro At different time, growth of LM in BHI liquid culture medium containing different concentrations of XL-184 has no difference.LM growth curves of the experimental group are similar to that of the control group in shape.2. The invasion rate of pathogenic bacteria LM on Caco-2 cells and HBMECs is higher than that of DH5a, BL21(DE3).For Caco-cells, LM invasion rate is about 0.168%, higher than that of the control group DH5 alpha 0.019%(P=0.000) and BL21 (DE3) 0.023%(P=0.000).For the HBMEC, LM invasion rate is about 0.129%, higher than that of the control group DH5 alpha 0.024%(P=0.000) and BL21 (DE3) 0.027% (P=0.000).3. C-Met blocking agents can reduce the invasion rates of LM to Caco-2 cells and HBMECsThe invasion rates of the experimental group are lower than the control group (P=0.000). Along with the increase of XL-184 concentrations, the invasion rate of LM to Caco-2 and HBMEC decrease. The invasion rate of LM to Caco-2 cells reduced after incubated with same doses of XL-184 for 1,2,6,12 h. The invasion rates of LM to HBMEC reduced after incubated with XL-184 for 1,2,6 h. With the increased doses of 17-AAG, invasion rates of LM to Caco-2 cells reduce. After incubated with 17-AAG for 1 h, invasion rates of LM to Caco-2 cells reduce.4. The combined effect of XL-184 and ampicillin is better than XL-184 alone in suppressing invasion rates of LM to Caco-2 and HBMECsInvasion rates of LM to Caco-2 cells in experimental group are lower than that of the control group (P0～1<0.01, P5<0.05), after incubated with different doses of XL-184 which respectively combined with AMP of 50 μg/ml for 24 h. With the increasing doses of XL-184, invasion rates of LM on Caco-2 cell decrease.Invasion rates of LM to HBMEC in the experimental group are lower than that of the control group (P<0.01), after incubated with different doses of AMP which respectively combined with 0.1μm XL-184 for 24 h. With the increasing doses of AMP, invasion rates of LM on Caco-2 cells decrease.Invasion rates of LM to HBMEC in experimental group are lower than that of the control group (P=0.000), after incubated with different doses of XL-184 which respectively combined with 50 μg/ml AMP for 24 h. With the increasing doses of XL-184, invasion rates of LM to HBMEC decrease.Invasion rates of LM to HBMEC in the experimental group are lower than that of the control group (P= 0.000), after incubated with different doses of AMP which respectively with 0.1 μm XL-184 for 24 h. With the increasing doses of AMP, invasion rates of LM to HBMEC decrease.5. The combined effect of 17-AAG and ampicillin is better than 17-AAG alone in suppressing invasion rates of LM to Caco-2 cellsThe invasion rates of LM to Caco-2 in the experimental group is lower than that of the control group (P0=0.000, P50～100<0.05, P250～500<0.01), after Caco-2 cells are incubated with different concentrations of 17-AAG which respectively combined with 50 μg/ml Amp for 24 h. With the increasing doses of 17-AAG, invasion rates of LM to Caco-2 cells decreased.The invasion rates of LM to Caco-2 cells in the experimental group is lower than that of the control group (P0=0.000, P5<0.01, P25～50<0.05, P100>0.05), after cells were incubated with various doses of AMP which respectively combined with 50 nM 17-AAG for 24 h. With the increasing doses of AMP, the invasion rates of LM to Caco-2 were decrease.6. XL-184 protects the permeability of Caco-2 cells and HBMECsThe decreased TEER value of Caco-2 monolayer is suppressed with the incubation of increasing XL-184 concentrations. HRP concentrations and the quantities of LM in the lower chamber also lowered (P=0.000). At the same time, with the extension of time that LM invades Caco-2 cells, the decrease of TEER values aggravate, the concentration of HRP and the number of LM in the lower chamber increase.The decreased TEER value of HBMEC monolayer is suppressed with the incubation of increasing XL-184 concentrations (P<0.05). HRP concentrations and the quantities of LM in the lower chamber also lowered (P<0.01). At the same time, with the extension of time that LM invades HBMEC, the decrease of TEER values aggravate, the concentration of HRP and the number of LM in the lower chamber increase.7.17-AAG protects the permeability of Caco-2 cellsThe decreased TEER value of Caco-2 monolayer is suppressed with the incubation of increasing 17-AAG concentrations (P<0.05). HRP concentrations (P=0.000) and the quantities of LM (P<0.05) in the lower chamber also lowered. At the same time, with the extension of time that LM invades Caco-2, the decrease of TEER values aggravate, the concentration of HRP and the number of LM in the lower chamber increase.8. XL-184 can lower the releasing level of LDH from Caco-2 cells and HBMECsAfter incubation of XL-184 with 1 and 24 h, LDH releasing rate of Caco-2 cells in the experimental group is lower than that of the control group. With higher concentrations of XL-184, the releasing rates of LDH decrease more obviously. (1h:P,<0.05, P2.5-10<0.01), (24h:P0.1～10<0.01)After incubation of XL-184 with 1 and 24 h, LDH releasing rate of HBMEC in the experimental group is lower than the control group. With higher concentrations of XL-184, the releasing rates of LDH decrease more obviously. (1h:P0.5-10<0.01), (24h: P0.5-10=0.000)9.17-AAG can lower the releasing level of LDH from Caco-2 cellsAfter incubation with 17-AAG for 1 and 24 h, LDH releasing rate of Caco-2 in the experimental group is lower than the control group. With higher concentrations of 17-AAG, the releasing rates of LDH decrease more obviously. (1h:P1000<0.001,24 h: P250～1000<0.001)10. XL-184 can lower the mortality rates of Caco-2 cells and HBMECs induced by LMCaco-2 cells are incubated with XL-184 in advance. The results show that cell survival rates in experimental groups are different from each other. When compared with the control group, the survival rates of the experimental groups are higher. (P<0.01)HBMEC are incubated with XL-184 in advance. The results show that cell survival rates in experimental groups are different from each other. When compared with the control group, the survival rates of the experimental groups are higher. (P<0.01)11.17-AAG can lower the mortality rates of Caco-2 cells induced by LMCaco-2 cells are incubated with 17-AAG in advance. The results show that cell survival rates in experimental groups are different from each other. When compared with the control group, the survival rates of the experimental groups are higher. (P=0.000)Conclusion1. C-Met blocking agent can not inhibit the growth of LM in vitro.2. C-Met blocking agents can reduce the invasion rates of LM to cells3. The combination of C-Met blocking agent and the penicillin antibiotics can effectively reduce the invasion rates of LM to cell.4. By reducing the invasion rates of LM to cells, c-Met blocking agents can inhibit the harmful effects of LM on the cells.5. By reducing the harmful effects induced by LM, c-Met blocking agents improve the viable rates of cells... |
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