Influence Of Baicalin On NADPH Oxidase-Mediated Inflammatary Response

ObjectiveAtherosclerosis (AS) is "The No.1Killer" causing human death at the moment, the pathological basis of many cardio-cerebrovascular diseases, and the pathogenesis, prevention and treatment of AS is always an important research topic in the field of medicine. In1999, Ross put forward the concept of "AS is an inflammatary disease", and points out that AS is a kind of inflammatory disease, not simply caused by fat deposition, induced a chronic inflammation by all sorts of damage through the release of cells factors, and chronic inflammation mediates the occurrence and development of the entire process. In recently, the relationship between Gram-negative bacteria infection and lipopolysaccharide(LPS), the important component of bacterium cell walls, and AS become a research hot spot. Macrophages play an important role in the development and occurrence of AS, study found that activated macrophages release complex of Calgranulin (S100) A8and Calgranulin A9(S100A8/A9) activates Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (NOX) mediated inflammatory response was related to inflammatory diseases such as AS. So by inhibiting macrophages’NOX-mediated inflammatory response can be a direction of prevent and therapy of AS. Based on the previous studies of our topic group, to further explore the resistance mechanism role of baicalin against AS. This study aims to explore:(1) Influence of baicalin and LPS on produce of S100A8/A9of macrophages;(2) Influence of baicalin and LPS on NADPH oxidase (NOX) gene expression of macrophages. MethodsThis research is mainly in vitro experiments, including the following contents:1Culture macrophage cells line RAW264.7in vitroInoculate RAW264.7into25cm2culture flask, use Dulbecco’s Modified Eagle Medium (DMEM)(Contain10%fetal bovine serum,100U/mL penicillin and100μg/mL streptomycin), and culture in incubator with5%volume fraction of CO2and37℃. When cells grow to80%cytomixis, use0.25%trypsin-EDTA for digestion, passage.2Influence of baicalin on RAW264.7’s proliferationInoculate growth logarithm period RAW264.75×104/mL100μL/hole in96holes tissue culture plate. When cells grow to single-desk covered with the hole, stimulate by medicine. Add100μL culture medium with concentration range baicalin (10,20,40,80,160,320,640,1280μg/mL) as baicalin group, and set up normal cells group without any stimulation, blank group (fill with sterile phosphate buffered saline, PBS),8holes/group. Cultured in incubator with5%volume fraction of CO2and37℃for24,48,72, and96h (Replace fresh medium every24h), use3-(4,5)-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium dromide (MTT) method to measure the oplical density (OD) of RAW264.7/hole by microplate reader in wavelength490nm.3Influence of baicalin and LPS on produce inflammation factors S100A8/A9in RAW264.7Digest growth good state cells by0.25%trypsin-EDTA, Trypan Blue Dye Exclusion Method appraises rate of cells living≥95%, adjust cells concentration to1×106/mL by DMEM, inoculate RAW264.71mL/hole in12holes tissue culture plate. When cells grow to grow to80%cytomixis, stimulate by medicine and culture.3.1Influence of LPS on produce of S100A8/A9in RAW264.7Add concentration range LPS (final concentration is0.1,1,10μg/mL), and set up normal cells group without any stimulation to stimulate,8holes/group. Cultured in incubator with5%volume fraction of CO2and37℃for4,8,12,16,20,24h, collect supernatant of treatment groups, use S100A8/A9Enzyme Linked Immunosorbent Assay (ELISA) Kit to measure content of cells factor by ELISA Method by microplate reader in wavelength450nm.3.2Influence of baicalin on produce of S100A8/A9in LPS-stimulated RAW264.7 The experiment groups are as follows:normal cell group, the model group, baicalin group, baicalinX LPS group. Add LPS (final concentration is1μg/mL) as model group, concentration range baicalin (final concentration is10,50,100μg/mL) pretreat1h as baicalin group, then add LPS(final concentration is1u g/mL) as baicalin×LPS group, and set up normal cells group without any stimulation,8holes/group. Cultured in incubator with5%volume fraction of C02and37℃for12h, collect supernatant of treatment groups, use S100A8/A9ELISA Kit to measure content of cells factor by ELISA Method by microplate reader in wavelength450nm.4Influence of baicalin and LPS on expression of NOX gp91phox (NOX2) mRNA in RAW264.7Inoculate growth logarithm period1×106/mL RAW264.7in12holes tissue culture plate. When cells grow to grow to80%cytomixis, stimulate by medicine and culture.4.1Influence of LPS on expression of NOX2mRNA in RAW264.7Add concentration range LPS (final concentration is0.1,1,10μg/mL), and set up normal cells group without any stimulation to stimulate,4holes/group. Cultured in incubator with5%volume fraction of CO2and37℃for4,8,12,16,20,24h, use TRIzol Method to extract total cells RNA.4.2Influence of baicalin on expression of NOX2mRNA in LPS-stimulated RAW264.7The experiment groups are as follows:normal cell group, the model group, baicalin group, baicalin×LPS group. Add LPS (final concentration is1μg/mL) as model group, concentration range baicalin (final concentration is10,50,100μg/mL) pretreat1h as baicalin group, then add LPS (final concentration is1μg/mL) as baicalin×LPS group, and set up normal cells group without any stimulation,4holes/group. Cultured in incubator with5%volume fraction of CO2and37℃for12h, use TRIzol Method to extract total cells RNA.Use Reverse Transcriptase-Polymerase chain Reaction (RT-PCR) Method to measure the expression of NOX2mRNA. Reverse Transcriptase use total RNA as template, Oligo dT-Adaptoras primer to reverse transcriptase synthetize cDNA, PCR use specific primer amplification target gene NOX2and reference gene GAPDH. See Table1. PCR products run in1%agarose gel electrophoresis, use Quantity One software to analysis the PCR products’Grey Values of of target gene NOX2and reference gene GAPDH, use the ratio of the Grey Values of target gene and reference gene the relative expression of target gene mRNA.Results1Cultured macrophage cells line RAW264.7in vitroObserved in inverted microscope, RAW264.7is adherent cells, the shape is round or irregular form, has pseudopods and typical "flagstone" closely set arrangement.2Influence of baicalin on RAW264.7’s prolferationInfluence of baicalin on RAW264.7’s proliferation, reviews a dependence on stimulated concentration and time of baicalin. Calculated24,48,72,96h half inhibitory concentration (IC50) results are1057.772,475.265,223.522.132.054μg/mL。For understand toxicity of baicalin and provides the basis for select safe dosage. This study choice baicalin stimulate concentration and time within1057.772ug/mL and24h.Mutiple linear regression of stimulated time, concentration of baicalin and OD values, regression equations:y=1.083-0.050x1-0.064x2,y is OD values, X(?) is stimulated time of baicalin, X2is concentration of baicalin, stimulated time, concentration of baicalin and OD values has a significant negative correlation (P<0.05).3Influence of baicalin and LPS on produce inflammation factors S100A8/A9in RAW264.73.1Influence of LPS on produce of S100A8/A9in RAW264.7In normal circumstances the culture supernatant of RAW264.7has little produce of S100A8/A9. Compared with normal group, LPS stimulation can promote produce of S100A8/A9(P<0.05), and reviews a dependence on stimulated concentration and time of LPS. Produce of S100A8/A9reach a peak value when LPS stimulate concentration and time is1μg/mL and12h, so choose1μg/mL,12h as LPS stimulate concentration and time.Mutiple linear regression of stimulated time, concentration of LPS and produce of S100A8/A9, regression equations: y=587.399+132.183x,+1068.888X2, y is produce of S100A8/A9, x1is stimulated time of LPS, x2is concentration of LPS, stimulated time, concentration of LPS and produce of S100A8/A9has a significant positive correlation (P<0.05).3.2Influence of baicalin on produce of S100A8/A9in LPS-stimulated RAW264.7Compared with normal group, baicalin(10、50、100μ g/mL) stimulated group has no statistical significance on produce of S100A8/A9(P>0.05).1μg/mL LPS group can significantly promote produce of S100A8/A9(P<0.001). In10-100μg/mL concentration range, baicalin pretreatment groups, compared with the model group can effectively ihibitate produce of S100A8/A9induced by LPS (P<0.05、0.001), and reviews a dependence on stimulated concentration of baicalin.4Influence of baicalin and LPS on expression of N0X2mRNA in RAW264.74.1Influence of LPS on expression of N0X2mRNA in RAW264.7In normal circumstances the expression of N0X2gene in RAW264.7is very low. Compared with normal group, LPS stimulation can promote expression of NOX2mRNA (P<0.05), and reviews a dependence on stimulated concentration and time of LPS. Expression of NOX2reach a peak value when LPS stimulate concentration and time is1μg/mL and12h, so choose1μg/mL,12h as LPS stimulate concentration and time.Mutiple linear regression of stimulated time, concentration of LPS and expression of N0X2mRNA, regression equations: y=0.226+0.019x1+0.177x2, y is expression of N0X2mRNA, x1is stimulated time of LPS, x2is concentration of LPS, stimulated time, concentration of LPS and expression of NOX2mRNA has a positive correlation, and concentration of LPS and expression of N0X2mRNA has a statistical significance (P<0.05).4.2Influence of baicalin on expression of NOX2mRNA in LPS-stimulated RAW264.7Compared with normal group, baical in(10、50、100μg/mL) stimulated group has no statistical significance on expression of N0X2mRNA (P>0.05).1μg/mL LPS group can significantly promote expression of NOX2mRNA (P<0.001). In10-100μg/mL concentration range, baicalin pretreatment groups, compared with the model group can effectively ihibitate expression of NOX2mRNA induced by LPS (P<0.001), and reviews a dependence on stimulated concentration of baicalin.ConclusionsProliferation of macrophage reviews a dependence on stimulated concentration and time of baicalin, and reveals a mutiple linear regression negative correlation with the stimulated concentration and time of baicalin. Calculated24,48,72,96h IC50results are1057.772、475.265、223.522、132.054μg/mL. For understand toxicity of baicalin and provides the basis for select safe dosage. LPS stimulation can cause proliferation of macrophage, as baicalin has a significant inhibition on the proliferation of LPS stimulation, and dependends on drug concentration.LPS stimulation can induce macrophage activation, promote cells factor produce of S100A8/A9and raise expression of NOX2mRNA, and reveal mutiple linear regression a positive correlation with the stimulated concentration and time of LPS. Baicalin pretreated can significantly inhibit LPS stimulation caused mass produce of cells factor S100A8/A9by macrophage, reduce expression of NOX2mRNA, and dependends on drug concentration.The experimetanl results show that baicalin can inhibit the proliferation of macrophage, inhibit produce of cells factor S100A8/A9from over-expressed, reduce expression of NOX2mRNA to inhibit imflammation, initially clarify the anti-AS mechanism of baicalin from anti-inflammation standpoint, provides theory basis for the application of baicalin.This research discusses the action and mechanism of baicalin inhibit AS in cell proliferation, gene adjustment and inflammation factors by the method of vitro experiments.