| Background:Endometritis is a common gynecological disease,usually caused by bacterial infection,decreased immune function,estrogen level disorders and other factors.Among them,bacterial infection is the main cause of endometritis.Patients often show various clinical manifestations,such as abdominal pain,irregular menstrual cycle,Abnormal Uterine Bleeding,and even infertility.It is reported that the prevalence of endometritis in premenopausal women is as high as 72 percent,which seriously affects the normal work and life of female patients.At present,antibiotics are often used in clinical treatment of endometritis,but long-term use of antibiotics is easy to lead to bacterial resistance and liver and kidney toxicity.Therefore,there is an urgent need to develop new drugs to treat endometritis.Pyroptosis is a kind of inflammatory cell death,whose main effect is to induce cell necrosis by releasing inflammatory mediators.TLR4/NF-κB/NLRP3 signaling pathway is one of the classical pathways mediating pyrodeath.Studies have shown that inhibition of TLR4/NF-κB/NLRP3 signaling pathway can effectively protect the endometrial tissue of mice with LPS-induced endometritis,and achieve a good anti-inflammatory effect.Clinopodium Chinense(Benth.)O.Kuntze is one of the plant sources of"Duanxueliu".It has the effects of dispelling wind and clearing heat,detoxifying and detumescence,promoting blood circulation and stopping bleeding.Previous studies have shown that a large number of flavonoids in the plant can reduce uterine bleeding,inhibit uterine tissue inflammation and promote endometrial repair in AUB rats.At the same time,a variety of flavonoids,such as apigenin,hesperidin and naringin,were detected in the drug-containing serum of rats.In addition,flavonoids in the plants have good antibacterial effect,which can be used against the endometritis caused by Bacillus tuberculosis,which suggests that total flavonoids of C.chinense(TFC)has the effect of anti-endometritis,and is worth further study and development.The purpose of this study is to explore the anti-endometritis effect and mechanism of TFC in order to provide new ideas for the prevention and treatment of clinical endometritis and the development of drugs.Purpose:In vitro and in vivo experiments were conducted to investigate the effect and mechanism of TFC on anti-endometritis in mice by inhibiting TLR4/NF-κB/NLRP3 signaling pathway and pyroptosis.Methods:1.Protective effect of TFC by inhibiting TLR4/NF-κB/NLRP3 signaling pathway on LPS-induced endometritis in mice(1)5 mg/mL LPS solution 20 μL was injected into the uterine cavity to establish a mouse model of endometritis;(2)The pathological changes of mouse endometrium were observed by HE staining;(3)The MPO activity in the uterine tissue of mice in each group was determined by spectrophotometry;(4)The ultrastructure of mouse endometrial epithelial cells(MEECs)was observed by transmission electron microscopy(TEM);(5)The secretion levels of TNF-α,IL-18 and IL-1β in uterine tissues were determined by ELISA;(6)The mRNA expression levels of TNF-α,IL-18 and IL-1β in uterine tissues of mice in each group were detected by RT-PCR;(7)Western blotting detected the expression of TLR4,IKBα,p-IKBα,p65,p-p65,Caspase-1,ASC,NLRP3 and GSDMD in mouse uterine tissue.2.TFC alleviates pyroptosis of MEECs by inhibiting TLR4/NF-κB/NLRP3 signaling pathway(1)Primary isolation and culture of MEECs were performed by collagenase digestion and sieve purification method and identification of MEECs by immunofluorescence method;(2)CCK8 method was used to select the optimal concentration and time of LPS on MEECs;(3)CCK8 method was used to screen the safe concentration of TFC drug-containing serum;(4)The effective concentration of serum containing TFC was screened by CCK8 method;(5)Morphological changes of MEECs were observed by inverted microscope;(6)Pyroptosis of MEECs was observed by Hoechst/PI fluorescence staining;(7)The ultrastructure of LPS damaged MEECs was observed by scanning electron microscope;(8)The nuclear entry of NF-κB p65 in MEECs was observed by immunofluorescence method;(9)The levels of inflammatory cytokines IL-1β and IL-18 in the supernatant of MEECs were determined by ELISA;(10)The protein expressions of TLR4,IKBα,p-IKBα,p65,p-p65,Caspase-1,ASC,NLRP3 and GSDMD in MEECs were detected by WB.3.The TFC inhibits the NLRP3 inflammasome activated by Nigericin to alleviate the pyroptosis of MEECs(1)NLRP3 expression in MEECs was detected by immunofluorescence method;(2)The levels of inflammatory cytokines IL-1β and IL-18 in the supernatant of MEECs were determined by ELISA;(3)The formation of ASC spots was observed by immunofluorescence method;(4)The expressions of NLRP3,Caspase-1,ASC and GSDMD in MEECs were detected by WB.Results:1.Protective effect of TFC by inhibiting TLR4/NF-κB/NLRP3 signaling pathway on LPS-induced endometritis in mice(1)Compared with the normal group,the serous surface of the uterine tissue of mice in LPS group showed hyperemia and swelling,and more serious inflammatory pathological changes.After treatment with TFC and FKQJC,the pathological changes of uterine tissue swelling and congestion were significantly improved.The endometrial cells were arranged neatly,and the tissue structure was complete and clear;(2)Compared with normal group,the activity of MPO in uterine tissue of mice was significantly increased after LPS treatment(P<0.01);Compared with model group,MPO activity in uterine tissue was significantly decreased in TFC medium dose,high dose and positive drug FKQJC group(P<0.05);(3)Transmission electron microscopy(TEM)showed that mouse MEECs showed smooth nuclear membrane,clear nucleoli,clear desmosomes and complete tight junctions between endometrial epithelial cells.After the treatment of LPS,the structure of MEECs was seriously changed,the nuclear membrane space was widened,some of them disappeared.The TFC and FKQJC groups significantly improved the damage of MEECs,the morphology of intracellular organelles tended to be normal,and the tight connections between cells were repaired;(4)Compared with normal group,the levels of TNF-α,IL-1β and IL-18 in LPS group were significantly increased(P<0.01);Compared with LPS group,The levels of inflammatory cytokines were significantly decreased after treatment with TFC and FKQJC;(5)Compared with normal group,mRNA expression levels of TNF-α,IL-1β and IL18 in uterine tissues of mice in LPS group were significantly up-regulated.Compared with LPS group,the expression levels of inflammatory factors TNF-α,IL-1β and IL-18 in TFC group and FKQJC group were significantly decreased;(6)Western blotting results showed that compared with LPS group,TFC could significantly down-regulate the expressions of TLR4,p-IKBα,p-p65,Caspase-1,ASC,NLRP3 and GSDMD.2.TFC alleviates pyroptosis of MEECs by inhibiting TLR4/NF-κB/NLRP3 signaling pathway(1)After primary isolation and culture of MEECs,the expression of epithelial marker protein keratin was identified by immunofluorescence method,and the positive rate of keratin was about 95%;(2)CCK8 assay showed that 1 μg/mL LPS treated MEECs for 24 h was the optimal concentration and time for the model,and 20%TFC medicated serum could significantly enhance the viability of LPS damaged MEECs;(3)Compared with the control group,2.5%to 20%of the serum containing TFC had no adverse effects on normal culture of MEECs;(4)Compared with LPS group,cell viability was improved in 2.5%,5%,10%and 20%TFC drug-containing serum groups(P<0.01),and the effect was most significant in 20%TFC drug-containing serum group;(5)Compared with the normal control group,the number of cells in LPS group was reduced,the cells had varying degrees of shrinkage,and a small number of cell fragments could be seen in the visual field.Compared with LPS group,TAK-242 group and 20%TFC drug-containing serum group,the degree of cell shrinkage and deformation was improved,the number of cells increased,and the cell morphology basically returned to normal;(6)Compared with normal control group,LPS group significantly increased the number of MEECs PI-positive cells and necrotic cells;Compared with LPS group,the red fluorescence of cells in TFC drug-containing serum group,TAK-242 group and TFC drug-containing serum+TAK-242 group was decreased,the number of PI positive cells was significantly decreased,and the number of necrotic cells was decreased(P<0.05);(7)The surface of the cell membrane in the normal control group was intact,and there were almost no pathological cell membrane pores.In LPS group,a large number of pores were formed on the surface of MEECs cell membrane,the number of microvilli was sharply reduced,and free cell matrix was exserted.Compared with LPS group,the morphology of cells in TFC drug-containing serum group and TAK-242 group was significantly improved,the number of membrane pores was significantly reduced;(8)Immunofluorescence results showed that the number of NF-κB p65 into the nucleus increased in LPS group,and both TFC drug-containing serum and TAK-242 could block p65 into the nucleus induced by LPS,and significantly reduce the content of p65 in the nucleus;(9)Compared with normal control group,the levels of cell secretion of IL-18 and IL-1β in LPS group were significantly increased(P<0.01);the secretion levels of inflammatory factors in TFC drug-containing serum group and TAK-242 group were significantly decreased(P<0.01);(10)Western blotting results showed that the expression of TLR4,p-IKBα,p-p65,Caspase-1,ASC,NLRP3 and GSDMD in MEECs could be significantly down-regulated by TFC.3.The TFC inhibits the NLRP3 inflammasome activated by Nigericin to alleviate the pyroptosis of MEECs(1)Compared with the normal control group,the expression of NLRP3 has increased to different degrees after cells stimulated by LPS and LPS+Nigericin.Compared with the LPS+Nigericin group,the expression of NLRP3 in the TFC medicated serum group was significantly reduced(P<0.01);(2)Compared with the normal control group,the LPS+Nigericin group has nigericin significantly increased the secretion levels of IL-18 and IL-1β(P<0.01);Compared with the LPS+Nigericin group,the TFC medicated serum group significantly reduced the secretion levels of IL-18 and IL-1β(P<0.01);(3)ASC fluorescence results showed that a small number of ASC spots were scattered around the cells after LPS was applied to MEECs sensitization.After the application of LPS and Nigericin on cells,most of the cells have formed granular spots around them.However,the TFC drug-containing serum group could significantly reduce the ASC spots around MEECs;(4)Western blotting results showed that compared with the LPS+Nigericin group,TFC obviously down-regulated the expressions of Caspase-1,ASC,NLRP3 and GSDMD in MEECs.Conclusion:TFC against endometritis in mice by alleviating pyroptosis of MEECs through inhibiting the TLR4/NF-KB/NLRP3 signaling pathway. |
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