| Mastitis is an important disease that restricts the development of the cow industry all over the world.The disease is mainly caused by the adhesion of pathogenic microorganisms(bacteria,fungi,mycoplasma,etc.)to the breast tissue.Therefore,antibiotic therapy is the main treatment for the disease which increases microorganisms antibiotic resistance easily,and antibiotic residues seriously threaten human health.Traditional Chinese medicine has low toxicity and side effects that has a good prospect for the clinical treatment of mastitis.Indirubin is a major active ingredient of traditional Chinese medicine such as indigo and Daqingye.It has anti-inflammatory and anti-tumor effects,but its clinical application is limited due to its poor water solubility and poorly absorbed in oral manner.6-Bromo-indirubin-3'-oxime(BIO,also known as 6BIO)is one of the derivatives of indirubin.Its anticancer effect is similar to that of indirubin,and its water solubility is better than that of indirubin.Studies have shown that BIO plays an important role in the treatment of breast and bladder cancers,but its therapeutic effect on mastitis has not been reported.In order to study the role of BIO in the treatment of mastitis,this study constructed mastitis models in vitro and in vivo in mice.The effect of BIO on mastitis was assessed by studying level changes in pro-inflammatory and anti-inflammatory cytokines after BIO intervention.By further testing the regulation of BIO on TLR4-NF-?B and TLR4-MAPK pathways,the regulatory mechanism of BIO on mastitis was determined.The apoptosis rate and apoptosis-related signal pathway changes were measured to determine the mechanism of BIO inhibition of mammary epithelial cell apoptosis during inflammation.This provided a theoretical basis for BIO to be used in clinical prevention and control of mastitis in cows.The main findings are as follows: 1.Determination of BIO concentrationMammary epithelium is a natural barrier of the mammary gland.Pathogenic microorganisms or their secreted toxins can cause loss of barrier function by damaging mammary epithelial cells and cause mammary gland damage.Mammary epithelial cells is a perfect model in vitro to study the effects of drugs on the mammary gland.In this study,the BIO concentrations of 1,5,25,50,100,200,500,and 1000 nM were used to conduct MTT assays.Different concentrations of BIO were used to challenge mouse mammary epithelial cells(MMECs).The results showed that concentrations of BIO were 1,5,25,and 50 nM had no effect on the growth of MMECs.Subsequently,the effect of the above concentrations of BIO on the transcription levels of pro-inflammatory cytokines IL-6,IL-1? and TNF-? in MMECs was examined by qRT-PCR.The results showed that the concentration of BIO were 5,25,and 50 nM could significantly inhibite the mRNA expression of IL-6 and IL-1? and TNF-?(p<0.001),and the inhibitory effect was most significant when the BIO concentration reached 50 nM,while 1 nM of BIO had no effect on pro-inflammatory cytokine levels.Therefore,BIO concentrations of 5,25,and 50 nM were selected for follow-up studies.2.BIO inhibits LPS-induced MMECs inflammatory responseTo investigate the effect of BIO on LPS-induced MMECs inflammation,MMECs were pretreated with different concentrations of BIO for 1 h and then stimulated with LPS.COX-2,IL-6,IL-1?,TNF-? and IL-10 were used to determine the effect of BIO on the LPS-induced inflammatory response of MMECs with testing their transcription levels and protein levels.The results of qRT-PCR and Western Blot showed that BIO at concentrations of 5,25,and 50 nM significantly reduced the LPS-induced expression of COX-2 both in transcription and protein levels(p<0.001)in a dose-dependent manner.The most significantly inhibitory effect of BIO on IL-6,IL-1? and TNF-? was 50 nM.On the other hand,BIO could dose-dependently promoted the expression of the anti-inflammatory cytokine IL-10.The above results indicate that BIO dose-dependently down-regulates the expression of COX-2,IL-6,IL-1?,TNF-? and up-regulates the expression of IL-10 to inhibit the LPS-induced inflammatory response of MMECs.3.BIO inhibits LPS-induced damage of mouse mammary glandsTo explore whether BIO has a regulatory effect on mouse mammary lesions caused by LPS,Kunming mice from 5-7 days postpartum were used as model animals.Dexamethasone was used as a positive control,blank group and DMSO were used as negative controls,and BIO concentrations were used.5,25,and 50 ?M.The results showed that all concentrations of BIO could reduce LPS-induced mammary gland swelling and congestion,reduce inflammatory exudation in the tissue,and protect the integrity of mammary gland structure.The scores of mammary inflammatory lesions in the BIO-treated group were significantly lower than those in the LPS group(p<0.001),indicating that BIO has a good protective effect on mouse mammary glands.In addition,BIO can reduce the expression of IL-6,IL-1?,TNF-? and MPO in tissue homogenate and serum.The down-regulation of BIO at a concentration of 50 ?M is most significant(p<0.001).The above results indicate that BIO has a significant protective effect on LPS-induced mammary gland injury,and this protective effect has a certain dose-dependence at the selected concentration,and 50 ?M has the best protection effect.4.BIO inhibits TLR4-NF-?B and TLR4-MAPKs activity in MMECsTo further study the mechanism of BIO inhibiting LPS-induced mouse mammary injury,we studied the effect of BIO on TLR4-NF-?B and TLR4-MAPKs,which play a key role in innate immunity.Effect of BIO on TLR4: In order to further study the role of BIO on TLR4,we used TAK-242 to inhibit TLR4 activity and observed changes in transcription levels of TNF-?.The results showed that TAK-242 inhibited TLR4 activity.The phenomenon of dose-dependent inhibition of TLR4 and TNF-? transcription disappeared.At the same time,qRT-PCR and Western Blot showed that BIO could significantly inhibit TLR4 mRNA expression and protein expression(p<0.001).It was shown that BIO can inhibit LPS-induced inflammatory damage by inhibiting TLR4 activity.The effect of BIO on NF-?B activity: NF-?B is an important nuclear transcription factor,and it is a key node for regulating innate immunity.To study the effect of BIO on it,we used qRT-PCR,Western Blot,and immunofluorescence to test the levels of phosphorylation,and nuclear entry.The results showed that different concentrations of BIO treatment can significantly down-regulate the expression of p65 mRNA(p<0.001),while all different concentrations of BIO can significantly inhibit the phosphorylation of p65,indicating that BIO can inhibit transcription and phosphorylation of p65,thereby inhibiting the activity of NF-?B and exerting anti-inflammatory effects.Effects of BIO on MAPKs: The effect of BIO on the three major members of the MAPK family,JNK,ERK,and p38,was investigated using qRT-PCR combined with Western Blot.The results showed that BIO could inhibit the expression of JNK,ERK,p38 transcription and protein levels,and the inhibitory effect of BIO on MAPKs showed a dose-dependent manner,and the inhibitory effect was most significant when BIO concentration was 50 nM(p<0.001).These results indicate that BIO can control the inflammatory response induced by LPS by inhibiting the activity of TLR4-NF-?B and TLR4-MAPKs in MMECs.5.BIO inhibits LPS-induced apoptosis of MMECs In the treatment of mastitis,in addition to eliminating the harmful factors of breast damage,anti-bacterial anti-inflammatory,improve the body immunity,we should protect the integrity of breast tissue,reduce cell apoptosis,accelerate the recovery of breast tissue.To study the effect of BIO on apoptosis of MMECs,we collected cells from different treatment groups(BIO treatment at 5,25,and 50 nM),analyzed the apoptotic rate by flow cytometry,and used qRT-PCR and Western Blot against Bak.Dynamic monitoring of changes in mRNA and protein levels of Bax,Bcl-2,Bcl-xl,Caspase-3,and Caspase-8.The results showed that BIO can reduce the apoptosis rate of LPS-induced MMECs,inhibit the expression of apoptosis genes Bak and Bax and the expression of apoptosis-related proteins caspase3 and caspase8(p<0.001),promote the suppression of apoptosis genes Bcl-2 and Bcl-xl expression.It was shown that BIO regulates the activity of both Caspase3 by regulating the expression of Bax,Bak,Bcl-2,Bcl-xl and Caspase8 inhibits LPS-induced apoptosis of MMECs.In summary,this study focuses on the effects of BIO on LPS-induced mouse mastitis,demonstrating that BIO can down-regulate IL-6,IL-1?,TNF-?,COX-2 and MPO expression and upregulation of IL-10 expression by inhibiting TLR4-NF-?B and TLR4-MAPK pathway activity,At the same time,BIO can inhibit LPS-induced apoptosis of MMECs.Therefore,this study lays a theoretical foundation for the clinical treatment of cow mastitis in BIO. |
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