Study On The Anti-inflammatory Mechanism Of Catalpol By Inhibiting NF-κB/MAPK Signal Pathway And Its Effect On Endometritis

Endometritis in dairy cows is a frequent reproductive system disorder in postpartum dairy cows.Clinically,the decline of postpartum immunity caused by poor individual condition,the occurrence of microbial infection in postpartum reproductive tract,and poor postpartum nursing can cause endometritis in dairy cows,which can seriously lead to infertility in dairy cows.reduce the production performance and economic value of dairy cows.At present,the treatment of endometritis in dairy cows includes antimicrobial drugs and hormone drugs.Long-term and frequent use of antimicrobials and hormone drugs will lead to drug resistance and drug residues in milk and body.once dairy products and meat enter the consumer market,it will lead to food safety,environmental safety and harm to human health.Traditional Chinese medicine is a natural medicine,which has a long history of drug use in veterinary clinic.the use of traditional Chinese medicine prescription in the treatment of endometritis in dairy cows will not produce the problem of drug residue and drug resistance in dairy cows,but the dosage is large and the dose of active components is difficult to determine,the interaction mechanism between traditional Chinese medicine is not clear.The analysis and purification of the effective components in traditional Chinese medicine and the pharmacological mechanism and dosage of the effective components in traditional Chinese medicine can greatly improve the utilization rate of traditional Chinese medicine.The medicinal history of Chinese herbal medicine Rehmannia glutinosa is very long.It has the effects of clearing heat,stopping bleeding,nourishing yin and nourishing fluid,and a large number of its active ingredients have been widely studied.Catalpol is one of the effective components extracted from Rehmannia glutinosa.At present,animal models have proved that catalpol has protective and anti-inflammatory effects on lung,kidney and heart,but the anti-inflammatory pharmacological mechanism of endometrium has not been reported.In this experiment,RAW264.7 cell inflammation model and dairy cow endometrial epithelial cell(bEECs)inflammation model were established,and mouse endometrial inflammation model was established.Through the intervention of catalpol,ELISA,RT-qPCR,Western blot and cellular immunofluorescence detection methods were used to study the anti-inflammatory mechanism of catalpol through the classical inflammatory pathways NF-κB and MAPK,in order to provide material and theoretical basis for the follow-up treatment of dairy cow endometritis.The results showed that when 3mM,0.3mM,0.03mM,3uM and 0.3uM were added to RAW264.7 cell culture medium and cultured for 6h,12h and 24h respectively,CCK-8 showed that the cell proliferation rate was the fastest at 12h culture.Different culture time and different concentration of catalpol had no significant effect on the proliferation of RAW264.7 cells,and different concentrations of catalpol had no toxicity to RAW264.7 cells.In the experiment,catalpol and RAW264.7 cells at the concentration of 3mM,0.3mM and 0.03mM were cultured for 12 hours as the basic setting for the follow-up experiment.The inflammatory model of RAW264.7 cells stimulated by LPS was established and treated with catalpol.ELISA,RT-qPCR,Western blot and cellular immunofluorescence detection showed that catalpol could down-regulate the levels of inflammatory factors IL-1β,IL-6 and TNF-α secreted by RAW264.7 cells stimulated by LPS,and reduce the gene and protein expression of TLR4.Further studies showed that catalpol attenuated the NF-κB signal transduction in RAW264.7 inflammatory cells by inhibiting the degradation of IκBa and the expression of p-p65 into the nucleus,and inhibited the MAPK signal transduction in RAW264.7 inflammatory cells by down-regulating the phosphorylation levels of p38,ERK and JNK,and the inhibitory effect on the above results was more obvious with the increase of catalpol concentration.The inflammatory model of bEECs cells stimulated by LPS was established,and the concentration of catalpol of ImM,0.1mM and 0.01mM which had no significant effect on the proliferation of bEECs cells was selected as the intervention experiment,and the 12h with the fastest proliferation rate of cultured cells was used as the basis of the follow-up experiment.The experiment was pretreated with dexamethasone and catalpol for 1 hour,and then stimulated by LPS for 12 hours.The rats were divided into blank group,LPS control group(lug/mL),dexamethasone positive control group(0.5ug/mL)and catalpol intervention group(1mM,0.1mM,0.01mM).The culture supernatant and cells were collected,and the gene and protein expression were determined by ELISA,RT-qPCR and Western blot.The results showed that LPS could significantly stimulate the expression of inflammatory cytokines IL-1β,IL-6,TNF-α and chemokine CXCL8,CXCL5 in bEECs cells,while the expression of inflammatory cytokines and chemokines decreased significantly in bEECs group pretreated with 1mM,0.1mM and 0.01mM in a dose-dependent manner.Cellular immunofluorescence assay showed that the expression of p65 in bEECs cells pretreated with catalpol was significantly inhibited,and the transduction of TLR4/NF-κB inflammatory signal pathway was also inhibited in different degrees.The mouse model of endometritis and catalpol intervention model were established.LPS(1mg/kg)was injected into the bilateral uterine horn of mice for 24 hours,and then 1mg/kg,10 mg/kg and 100 mg/kg catalpol were injected intraperitoneally for 24 hours.ELISA,RT-qPCR,Western blot and cellular immunofluorescence were used to detect the expression of inflammatory factors,chemokine,TLR4/NF-κB signal pathway protein and MPO activity in uterine tissue of mice.The results showed that catalpol of different concentrations could decrease the activity of MPO in uterine tissue of mice.Decrease the gene expression levels of inflammatory factors IL-1β,IL-6,TNF-α in serum and chemokine CXCL8 and CXCL5 in uterine tissue,and inhibit the protein expression of TLR4/NF-κB signal pathway in uterine tissue of inflammatory mice.The results showed that catalpol could effectively improve the inflammatory state of endometrium,and the higher the concentration of catalpol,the more obvious its anti-inflammation.To sum up,catalpol inhibits TLR4 signal transduction in a dose-dependent manner,blocks the expression of NF-κB and MAPK signal pathways downstream of catalpol,and protects endometrium from inflammation by significantly reducing the activities of inflammatory cytokines IL-1β,IL-6,TNF-α and chemokine CXCL8,CXCL5 and MPO in uterine tissue.