Study On Anti-inflammation Effect And Anti-inflammatory Mechanism In LPS Induced Endometrial Epithelial Cells Of Palmatine

Endometritis is considered an acute or chronic inflammatory reaction because mammals fail to completely clear postpartum bacterial contaminants of the endometrium in time.It severely impairs the sound development of cows and other economic animal breeding industry.Currently,antibiotic,hormone,probiotics and traditional Chinese medicine are widely used to treat endometritis in clinic.Traditional Chinese medicine can resist bacterial infection,improve blood circulation and immune function of body,and enhance uterine contraction.Also there is no drug residues and toxic side.So the study of Chinese herbal medicine for the treatment of endometritis has became a research hotspot.Palmatine,an important traditional medicinal compound,has various biological activities usually used to treat jaundice,hypertension,dysentery,inflammation,and liver-related diseases.However there are few reports about the anti-inflammatory effects of palmatine on endometritis in veterinary.The aim of this study was to investigate the anti-inflammatory effects of palmatine on lipopolysaccharide?LPS?-induced inflammation in goat endometrial epithelial cells?g EECs?,and the possible molecular mechanisms,serial researches were carried out in this paper.1.Explore the cytotoxicity of palmatine on gEECsTo explore the cytotoxicity of palmatine on gEECs,goat endometrial epithelial cells?gEECs?were stimulated by different concentrations of palmatine,then the growth curve of cell was drew by MTT assay which could detect the cell proliferation.The cell growth state was observed by inverted microscope,and Wright-Giemsas taining was used to observe morphological changes of the cells while LDH test was used to check the membrane permeability of EECs.The results showed that the safe dose range of palmatine was less than 250?g/mL,and the morphology of the nucleus and cytolymph would not significantly change.While when the concentration was more than 250?g/mL,there will be a certain toxic effect on gEECs,and enhance the permeability of cells membrane.when the concentration was 10 to 100?g/mL,palmatine had a role in promoting proliferation of g EECs and in good condition.So low,medium and high?20,40 and 80?g/mL?concertration of palmatine were chosed to the subsequent experimental study.2.Palamtine modulated inflammatory cytokines in LPS-stimulated gEECS inflammation modelTo establish the inflammation model,gEECS were exposed to LPS?5?g/mL?for 12 h?LPS group?,then treated with Palmatine at various concentrations?80,40and20?g/mL?for additional 4,8,12,24and 36 h.The levels of TNF-?,IL-1?,IL-6,PGE₂,MMP-9,MMP-2 and IL-10 in the supernatant were quantified using ELISA kits,and the level of NO was spectrophotometrically determined by assaying thenitrite.The result showed that LPS significantly increased the TNF-?,IL-1?,IL-6 and IL-10 levels in LPS group compared with the control group?P<0.05?.Palmatine possesses anti-inflammatory bioactivities in LPS-induced gEECs inflammation model,and regulates the levels of cytokines.On one hand,palmatine treatment significantly inhibited the production of TNF-?,IL-6,IL-1?and NO?P<0.05?.On the other hand,palmatine significantly promoted the secretion of IL-10?P<0.05?.In addition,palmatine can protect the structural integrity of cells from inflammatory injury by inhibiting the production of MMP-2 and MMP-9.3.Effects of Palmatine on the signaling pathway of TLR4 in LPS-stimulated gEECSTo establish the inflammation model,gEECS were exposed to LPS?5?g/mL?for 12 h?LPS group?,then treated with palmatine at various concentrations?80,40 and 20?g/mL?for additional 8 h.RT-PCR and Western blotting were used to detect the expression of proteins in the TLR4 signaling pathway.The result showed that LPS significantly up-regulated gene expression of IL-1?,TLR4,CD14 and TICAM compared with control group?P<0.05?.Palamine can intensively supress the gene expressions of IL-1?,TLR4,CD14,NF-?Bp65 and TICAM,and the phosphorylation of NF-?Bp65.Noted that there was no distinct changes of MyD88 in either LPS group or palamine treatment groups?P>0.05?.Palmatine possesses anti-inflammatory bioactivities in LPS-induced gEECs inflammation model.The possible anti-inflammatory mechanism of palmatine was related to the inhibition of TRIF-dependent and NF-?B signaling pathways at the gene level.In summary,Palmatine possesses anti-inflammatory bioactivities in LPS-induced gEECs inflammation model,and regulates the levels of cytokines.The possible anti-inflammatory mechanism of palmatine was related to the inhibition of TRIF-dependent and NF-?B signaling pathways.