| Part I Correlation between Intestinal Flora and its Metabolites and Bone Cancer PainObjective To establish a mouse model of bone cancer pain and explore the changes of intestinal flora;To explore the effect of intestinal flora on bone pain.Methods SPF grade C57 BL / 6 mice were divided into normal control group(Group C),sham operation group(Group s),bone cancer pain group(group P),bone cancer pain + antibiotic group(PA group)and bone cancer pain+ flora transplantation group(PF group).Lewis lung cancer cells were used to establish the bone cancer pain model.The pain behavior was evaluated by observing the changes of general conditions of mice,measuring the number of spontaneous foot lifting(NSF)and mechanical foot retraction reflex threshold(pwmt).The tumor growth was observed by X-ray examination and he staining of femoral section,so as to judge whether the bone cancer pain model was successfully established;The fecal flora of mice in each group was analyzed by16 Sr RNA amplicon high-throughput sequencing technology;The short chain fatty acids in serum and brain were detected and analyzed by gas chromatography-mass spectrometry(GC-MS);The permeability of blood-brain barrier was measured by perfusion of Evans blue;The expressions of occludin and claudin-5 were detected by Western blot.Results Compared with the sham operation group,the pwmt of mice in BCP group decreased significantly and the NSF increased significantly at each time point after operation.X-ray examination showed that there was obvious bone destruction in the femur of BCP group,and the degree of destruction was positively correlated with the modeling time.HE staining of femur showed that the tissue lesions in the bone marrow cavity of the model group were more serious,the cavity was filled with large and deeply stained tumor cells,and there was obvious bleeding,and some cortex was damaged and damaged.16 Sr RNA showed that the occurrence of bone cancer pain reduced the abundance of intestinal flora in mice.The ratio of Firmicutes / Bacteroidetes(F / b)increased significantly;The relative abundance of Clostridium,lactobacilli and prevotellaceae decreased the most.The content of SCFAs in mouse brain tissue detected by GC-MS showed that the content of butyric acid in serum and brain tissue decreased after bone cancer pain.Conclution The intestinal flora of mice with bone cancer pain was disordered,and Clostridium,lactobacilli and prevotellaceae decreased significantly.Bone cancer pain reduces butyric acid production by affecting intestinal flora.The change of intestinal flora will affect bone cancer pain,and fecal bacteria transplantation(FMT)can reduce bone cancer pain.The permeability of blood-brain barrier is associated with bone cancer pain.Intestinal flora imbalance may affect pain by changing the permeability of blood-brain barrier.Part II Mechanism of Intestinal Flora Metabolite Butyric Acid Mediated Intestinal Brain Axis Affecting Bone Cancer PainObjective To investigate the mechanism of butyric acid regulating the permeability of blood-brain barrier and its effect on microglia polarization.Methods 1.The mice were divided into blank control group(Group C),bone cancer pain group(group P),bone cancer pain+antibiotic group(PA group),bone cancer pain+antibiotic+Clostridium butyricum group(PB group),bone cancer pain+antibiotic+sodium butyrate group(PNA group),PPAR-γ group Inhibitor group(CG group),bone cancer pain+PPAR-γ Inhibitor group(PG group),bone cancer pain +Clostridium butyricum+PPAR-γ Inhibitor group(PBG group),bone cancer pain+sodium butyrate+PPAR-γ Inhibitor group(PNG group).Permeability of blood-brain barrier was detected by perfusion of Evans blue.The expressions of Occludin and Claudin-5 were detected by Western blot.Transcriptome sequencing was used to detect the differentially expressed genes before and after the use of Clostridium butyricum.PPARγ/COX-2 expression was detected by Western blot.Immunofluorescence was used to detect Iba1 、TNF α、 PPAR γ and TLR4 expression.2.BV2 cell experiment in vitro: BV2 cells were divided into blank control group(control group),sodium butyrate treatment group(control+NA group),lipopolysaccharide treatment group(LPS group),LPS + sodium butyrate treatment group(LPS + NA group),LPS + sodium butyrate + GW9662 treatment group(LPS+NA+GW9662 group).The morphological changes of cells were observed under inverted phase contrast microscope;CCK-8 cell proliferation toxicity assay.The proportion of M1 and M2 microglia was detected by flow cytometry;Expression of PPARγ 、 TLR4 was detected by cellular immunofluorescence.Results Pain behavior test showed that cmpared with PB group,PWMT decreased significantly and NSF increased significantly in PBG group(P < 0.05).Compared with PNA group,PWMT decreased significantly and NSF increased significantly in PNG group(P < 0.05).Evans blue test showed that the permeability of blood-brain barrier in PBG group and PNG group was higher than that in PB group(P < 0.05).Western blot showed that the expressions of Occludin and Claudin-5 in PB group and PNA group were significantly higher than those in P group(P < 0.05).The protein expression of Occludin and Claudin-5 were significantly decreased in PBG group and PNG group(P < 0.05).The transcriptome screened out top20 differentially expressed genes,in which peroxisome proliferators activated receptors(PPARs)were enriched and scored higher in Clostridium butyricum group.Western blot showed that PPARγexpression in group P decreased and COX-2 expression increased significantly(P < 0.05).Compared with group P,PPARγ expression in PNA and Pb groups increased significantly(P < 0.05),and the COX-2 expression decreased(P <0.05).Compared with PB group,the expression of COX-2 in PBG group was significantly higher(P < 0.05).Compared with PNA group,the expression of COX-2 in PNG group was significantly higher(P < 0.05).Immunofluorescence showed that compared with group C,group P had TNF α The percentage of M1 microglia co labeled with Iba1 increased(P < 0.05).Compared with group P,after treatment with Clostridium butyricum,the percentage of cells co labeled with TNFα and Iba1 in the total Iba1 positive cells decreased,and the difference was statistically significant(P < 0.05).The percentage of cells co labeled with TNF α and Iba1 in PBG group in the total Iba1 positive cells increased,and the difference was statistically significant(P < 0.05).Compared with PNA group,the percentage of cells co labeled with TNF α and Iba1 in PNG group in the total Iba1 positive cells increased.immunofluorescence result display that,in Group P,the expression of PPARγ decreased,whereas TLR4 increased,the difference was statistically significant(P < 0.05).Compared with group P,the expression of PPARγ and TLR4 in Pb group and in PNA group increased,which was statistically significantly different(P < 0.05).After PPARγ antagonist GW9662 given,the expression of TLR4 in PBG group was significantly higher than that in PB group(P < 0.05).Compared with PNA group,the expression of TLR4 in PNG group was significantly higher(P < 0.05).3.Experimental part of BV2 cells in vitro: The results of inverted phase contrast optical microscope showed that the microglia in the control group were in a resting state,the cell body was slender,and the branches of slender processes were extended on both sides of the cell body.In LPS group,most cell bodies became larger and rounder,and some cell surface protrusions became thicker and shorter,showing a typical amoeba shape.The cell morphology was improved after pretreatment with sodium butyrate.CCK8 results showed that BV2 microglia treated with different concentrations of sodium butyrate(1.25mmol/L-10mmol/L)had no significant difference in cell activity compared with the blank control group.It showed that different concentrations of sodium butyrate had no significant effect on cell activity.The results of flow cytometry showed that M1/M2 in LPS group was higher than that in control group.Compared with LPS group,M1/M2 decreased in LPS+NA group.Compared with LPS+NA group,M1/M2 in LPS+NA+GW9662 group increased(P < 0.05).Immunofluorescence results showed that,compared with control group,TLR4 fluorescence intensity in LPS group increased significantly,while the PPARγfluorescence intensity decreased(P < 0.05).Pretreatment with sodium butyrate could reduce TLR4 and increase PPARγ fluorescence intensity(P < 0.05),but GW9662 could antagonize this effect.Conclusion Clostridium butyricum and sodium butyrate can inhibit bone cancer pain.Butyric acid can regulate bone cancer pain by regulating bloodbrain barrier permeability through intestinal brain axis,which is associated with butyric acid/PPARγ/COX-2 signal pathway.The decrease of pain threshold caused by bone cancer pain is closely related to central sensitization.Butyric acid can affect central sensitization by regulating the polarization of microglia,which is closely related to butyric acid/PPARγ/TLR4 signal pathway.Part III Effect of oxymatrine on bone cancer pain through intestinal floraObjective To explore the mechanism of oxymatrine on bone cancer pain by regulating intestinal flora.Methods 1.Mice were randomly divided into blank control group(Group C),sham operation group(Group S),bone cancer pain group(group P),Oxymatrine group(Group CO),sham operation + antibiotic group(group SO),bone cancer pain+Oxymatrine group(Group PO).The number of spontaneous foot lifting(NSF)and mechanical foot retraction reflex threshold(PWMT)were measured to evaluate the changes of pain behavior;The fecal flora of mice in each group was detected and analyzed by 16 Sr RNA technology and highthroughput sequencing technology;The short chain fatty acids in serum were detected and analyzed by GC-MS;The expression of short chain fatty acid transporter Slc5a8 protein in the brain of mice in each group was detected by immunohistochemistry;PPARγ/COX-2 expression was detected by Western blot.2.Experiment of BV2 cells in vitro : The cultured microglia were randomly divided into blank control group(control group),lipopolysaccharide pretreatment group(LPS + OMT group),sodium butyrate and Oxymatrine CO treatment group(LPS+NA+OMT group).CCK-8 cell proliferation toxicity test was used;The proportion of M1 and M2 microglia was detected by flow cytometry;PPAR was detected by cellular immunofluorescence technique γ、Expression of TLR4.Results Pain behavior test showed that PWMT in PO group was significantly higher than that in P group on the 4th day after model preparation(P < 0.05);Compared with group P,NSF in PO group decreased significantly on the 4th day(P < 0.05).16 Sr RNA showed that after intraperitoneal injection of oxymatrine,the relative abundance of Prevotellaceae,Clostridaceae and Trichospirillaceae in the intestine of bone cancer pain mice increased,while rumen bacilli and Desulfovibrio decreased significantly.The results of GC-MS showed that the concentration of serum butyric acid increased significantly on the 21 st day after bone cancer pain.Evans blue test showed that the permeability of blood-brain barrier in Po group was lower than that in P group(P < 0.05).Immunohistochemical results showed that the positive rate of Slc5a8 protein in group P was lower than that in group C;The positive rate of Slc5a8 protein in Po group was significantly higher than that in P group;Compared with group C,the positive rate of Slc5a8 protein in CO group was higher.Western blot showed that the expression level of PPARγ in group P was lower than that in group C(P<0.05)and the expression level of COX-2 increased significantly(P<0.05).Compared with group C,there was no significant difference in the expression levels of PPARγand COX-2 in CO group(P > 0.05).Compared with group P,the expression level of PPARγ in PO group increased and the expression level of COX-2 decreased(P < 0.05).CCK8 assay showed that Oxymatrine at different concentrations had no significant effect on cell activity.The results of flow cytometry showed that M1/M2 in LPS+OMT group was significantly lower than that in LPS group(P <0.05).Compared with LPS group,M1 / m in LPS + Na + OMT group decreased significantly(P < 0.05).Immunofluorescence showed that compared with LPS group,the expression of PPARγ in LPS + OMT group increased and the expression of TLR4 decreased.Compared with LPS group,the expression of PPARγ in LPS+NA+OMT group increased and the expression of TLR4 decreased.Conclusion Oxymatrine can relieve bone cancer pain.After intraperitoneal injection of oxymatrine,it will cause the changes of intestinal flora in mice with bone cancer pain,mainly reflected in the changes of F / B and bacterial abundance,and the increase of butyrate producing bacteria.The concentration of butyric acid in serum of mice with bone cancer pain increased.Oxymatrine through butyric acid / PPAR γ/ COX-2 signaling pathway mediates blood-brain barrier to regulate bone cancer pain;Oxymatrine can affect central sensitization by regulating the polarization of microglia γ/ TLR4 pathway is closely related. |
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