| Objective: In the early stage of the project team,Zhuang medicine Fu Yajing granule has develop new preparations,the previous research found that the granule is better to treat Sequelae of PID model of rats,it can improve the mixing of infection with mechanical damage of rats with Sequelae of PID,but its mechanism is unknown.The NF-?B is an important transcription factor present in the cytoplasm,immune and inflammatory responses in a large number of gene expression is affected by NF-? B regulation,including cytokines(such as TNF-?)?adhesion molecules(such as ICAM-1)and chemokines,etc,while involved in inflammation amplication and continuation of various enzymes expression(such as nitric oxide synthase NOS)has an important regulatory role,thereby affecting the body's inflammatory response.In this study,Sequelae of PID model of rats was caused by injection of a mixture of bacterium suspension into injured womb with mechanical injury method,and therapy by giving appropriate amount of Zhuang medicine Fu Yajing granule,by detecting the expression of TNF-??TL-10?NO?NOS?SOD?MDA?ICAM-1?NF-?B?NF-?BmRNA and ICAM-1 mRNA,to explore the mechanism of action about Zhuang medicine Fu Yajing granule treat Sequelae of PID model of rats,it provides experimental evidence for Zhuang medicine Jinmu granule clinical treatment of Sequelae of PID.Methods:(1)Preparation strong medicine woman Accor net new granule formulation according to Zhuang Fu Yajing net proportion of prescriptionsdrugs weighed herbs 10 Kg,plus 10 times 60% ethanol was extracted twice,each time for 2 hours and 300 mesh filter cloth,the filtrate was concentrated to a thick dip cream,condensed extract solid content was measured by adding an appropriate amount of soluble starch stir theory per g of dry extract containing raw herbs 2.33 g,14-16 mesh sieve granulation,80 ? blow dried,granulated 14 mesh screen,press Operators have to paste 21%,10 kg deserve extract 2.1kg,a dry plaster on original dose of medicine 2.33 g / g,should be added accessories 2.19 kg.After pressing the bag 12 g aliquots,equivalent to the original medicine.(2)Modeling: Sequelae of PID model of rats was caused by injection of a mixture of bacterium(escherichia coli,staphylococcus ratio of 1:1 was dissolved in sterile saline dubbed the concentration of 3×109cells/ml of the mixed)suspension into injured womb with mechanical injury method which is model group,To open the rat's abdomen without treatment and then stitch it as sham group.After the modeling section 1d,3d,5d,7d,14 d,21d,28 d take the model group,the sham group and the normal group of rats' blood and the uterus to do the blood routine examination and pathological section.By detecting the blood routine and pathological section to evaluation of statistical scores to determine how many days after building form Sequelae of PID model.(3)Coverage,treatment: After building Sequelae of PID model of rat,then the rats were randomly divided into several groups as follows: normal group,sham group,model group,Huahong particles positive group,dexamethasone positive group,Zhuang medicine Fu Yajing granule group dose of high,Zhuang medicine Fu Yajing granule group dose of middle,Zhuang medicine Fu Yajing granule group dose of lower.After modeling the rat to weight every other day,and to give corresponding drug and pure water to treatment in 15 days.In 15 d after lavage 1 hour,the anesthesia in rat,takethe rats' thymus,spleen and uterus,then weighing,and calculate the viscera coefficient values.A part of the whole blood to do the blood hemorheology and blood routine,another part of the blood to static centrifugal after take serum to test.The uterus tissue is divided into three parts,a part of uterus is soaking with 4% paraformaldehyde,for pathological and immunohistochemical,part of uterus add to a certain volume of physiological saline homogenate,take supernatant on standby,the last part of uterus put into the frozen in rapidly,and then put it in-80? refrigerator to standby.(4)Using WST-1 to detect Superoxide Dismutase(SOD)vitality in blood serum,using TBA to detect Methane Dicarboxylic Aldehyde(MDA)content in blood serum,using nitrate reductase to detect Nitric Oxide(NO)content in blood serum,using chemical colorimetry to detect Nitric Oxide Synthase(NOS)vitality in blood serum.(5)Using ELISA to detect interleukin-10(IL-10)and tumor necrosis factor-?(TNF-?)concentration alteration in tissue fluid.(6)Using immunohistochemical method to detect nuclear factor–?B(NF-?B)and intercellular adhesion molecule-1(ICAM-1)expression in rat uterus tissue.(7)Using reverse transcription polymerase chain reaction(RT-PCR)technique to detect nuclear factor-?B messenger RNA(NF-?B mRNA)and intercellular adhesion molecule-1 messenger RNA(ICAM-1mRNA)expression in rat uterus tissue.Results:(1)The Zhuang medicine Fu Yajing particles brownish yellow,slightly bitter taste,according to 12 g / packing bags,lot number: 20150726,stored in a desiccator and set aside.(2)In 14 d building model the number of leukocytes in blood serum in rats rise obviously(P<0.01),the number of neutrophils increased significantly(P<0.05),lymphocytes decreased significantly(P<0.05).Athological HEstaining were observed that the first 14 d intrauterine expansion chamber wall structural changes,the presence of inflammatory cell infiltration.After building model rats weight decline sharply,in about 5 days began to regain weight,for 14 days drug treatment,model group uterus dirty body ratio is bigger but not significant(P>0.05),the thymus and spleen were significantly smaller(P<0.01),the number of leukocytes in blood serum in rats rise obviously(P<0.01),the number of neutrophils increased significantly(P<0.01),lymphocytes decreased significantly(P <0.01),model group uterus anatomical morphology comprehensive score significantly higher(P < 0.05).It indicates that the formation of Sequelae of PID of disease models after the building in the 14 th day.(3)Compared with rats in normal group,SOD in blood serum in rats of model group reduces distinctly(P<0.01).MDA,NO and NOS rise obviously(P <0.05,P <0.01 or P <0.05).Compared with the model group,SOD activity of Zhuang medicine Fu Yajing granule group were significantly higher(P<0.01),MDA content of Zhuang medicine Fu Yajing granule group were decline vividly(P<0.05),NO content and NOS activity of Zhuang medicine Fu Yajing granule group were reduces distinctly(P <0.01).(4)Compared with rats uterine tissue fluid in normal group,the concentrations of IL-10 and TNF-? of model group significantly higher(P<0.01).Compared with the model group,the concentrations of IL-10 and TNF-? of positive group and Zhuang medicine Fu Yajing granule group were decline vividly(P <0.01).(5)Compared with rats uterine tissue in normal group,the immune expression of NF-?B?ICAM-1 of model group significantly higher(P <0.01).Compared with the model group,the immune expression of NF-?B of Zhuang medicine Fu Yajing granule group were decline vividly(P <0.01),the immune expression of ICAM-1 of Zhuang medicine Fu Yajing granule group of highdecline vividly(P<0.01),Zhuang medicine Fu Yajing granule group of middle and lower were reduces distinctly(P<0.05).(6)Compared with rats uterine tissue in normal group,NF-?B mRNA and ICAM-1 mRNA expression in the model group increased significantly(P<0.05).Compared with the model group,NF-?B mRNA expression and ICAM-1 mRNA expression in Zhuang medicine Fu Yajing granule group doses of lower reduces distinctly(P<0.05).Conclusion(s):(1)Preparation of simple Zhuang medicine Fu Yajing net new granule formulation is conducive to future industrial production.(2)In this study,plus mixed bacteria was established by the initial mechanical injury time Sequelae of PID disease in animal models for the formation of the first 14 d after modeling.(3)The mechanisms of result about Zhuang medicine Fu Yajing granule treatment Sequelae of PID model in rats,up-regulation the levels of SOD anti-inflammatory,down-regulation the levels of MDA?NO?NOS,IL-10?TNF-??NF-?B?ICAM-1?NF-?BmRNA?ICAM-1mRNA,to impover the damage of Sequelae of PID. |
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