Anti-inflammatory Effect And Mechanisms Of Indirubin In LPS-induced Mouse Mastitis

Bovine mastitis is a common and frequently-occurring disease in dairy cows.It is very damaging,overspending,and difficult to prevent and treat,severely hindered the development for dairy industry worldwide.Most of the cow mastitis was caused by pathogenic microorganism through adherence to breast tissue.More than 130 pathogenic microorganisms causing mastitis have been isolated and identified,which shows that approximately 80% bovine mastitis are caused by Escherichia coli,Staphylococcus aureus and Streptococcus.Although studies on prevention and treatment of bovine mastitis have been developed for more than 100 years,there is still no effective method to solve it.Antibiotic therapy is the prefered method for mastitis treatment around the world.However,with the long-term and even abuse use of antibiotic,microbial resistance and antibiotic residual are more and more common,gradually become a bottleneck to restrict the prevention and control on mastitis.The drug resistance of foodborne animal pathogens can be transmitted to humans through the food chain and drug resistance plasmid,which has caused unprecedented challenges and problems to human.Traditional Chinese medicine are natural herbs including many biologically active components.It has been increasingly favored by domestic and foreign experts and scholars,due to the widespread effects,no or fewer use drug resistance,lower toxicity,no or low residues,dual effects as drugs and nutritional agents,Indirubin belongs to indole compound extracted from indigo.Indirubin is one of the most effective components on traditional Chinese medicine,eg.indigo,Folium Isatidis,radix isatidis,etc..Indirubin plays a significant role on anti-leukemic,anti-bacterial,anti-inflammatory,neuroprotective,anti-virus and anti-parasites,and even be much more effective compared with antibiotics.It has a great potential to prevent and treat bovine mastitis,achieve green breeding and production without drug residues in animal products.Herefore,we examined a possibleindirubin effects on anti-mastitis.Here,from molecular,cell to organism level,we studied the preventions,treatments and mechanisms of indirubin to LPS induced mouse mastitis which laid the foundation for prevention and treatment of bovine mastitis.The main contents are as follows.1.Extraction and identification of mouse mammary epithelial cellsMMECs were aseptically extracted by trypsin and collagenase digested method from mouse mammary glands tissue,and the mice were pregnant for 18 to 20 days.The morphology of MMECs were round,oval or olive,which adhered to cell culture flask after 12 hours,and filled up T75 cell culture flask after 48 hours.When the cells are covered the flask,the cells present as paving stone-like.In Ceratin-18 labeled MMECs,single and bright green fluorescent can be detected by immunofluorescence(IF)and,cells grow well with a high purity.2.Determination of the challenge concentration of indirubin and lipopolysaccharideIn order to optimize the LPS challenge concentration,a series of concentration were set up to stimulate MMECs and,detected the m RNA expression of IL-1?,IL-6 and TNF-? by q RT-PCR.The results indicated that with the increasing concentration of LPS,the expression levels of three inflammatory factors increased contantly,and especially it is significant higher than unstimulated group at 1?g/m L LPS(p < 0.001).When compared with the level of 1?g/m L LPS,the rising trend of the three inflammatory factors slowed down in 2?g/m L LPS.We finally choose 1?g/m L as stimulation concentration in the following experiments.On the other hand,the cytotoxicity of indirubin was determined by MTT assay in the presence of 1-1000 n M indirubin,and then the data proved that low concentration of indirubin(1-250 n M)have no significant cytotoxicity to MMECs unless the concentration is higher than 250 n M.We also confirmed that MMECs viability shows no difference with or without LPS,as well as the processing order of LPS and indirubin.Furthermore,1-1000 n M indirubin were used to determine their effects to the LPS induced MMECs inflammation,and consequently,over 25 n M indirubin enable to significantly inhibited the expression of IL-1?,IL-6,and TNF-? in MMECs(p < 0.001).Thus,theconcentration of 25,50,and 100 n M indirubin were selected to study the effects on the prevention and treatment of MMECs inflammation.When come to animal models,the dose increased to 1000 times.3.The mechanism of indirubin treatment on LPS-induced inflammation in MMECs and mouse mastitisMMECs(1×106 cells)were seeded into six-well plates and grown until 80% confluent.MMECs were induced by LPS for 1h,added indirubin co-cultivated for another 24 h.ELISA and q RT-PCR were used to determine the expression level of IL-1?,IL-6 and TNF-? by LPS induced MMECs.The result showed that the expression level of pro-inflammatory cytokines was decreased by indirubin in a dose dependent manner,and significantly at 100 n M(p < 0.001).We subsequently detected the effect of indirubin on COX-2 via western blotting and q RT-PCR.The result revealed that both negative group and positive group showed no difference with blank group,while experimental group showed decreased expression level in a dose dependent manner compared with positive group.LPS was injected into the udder canal of postpartum 5-7d Kunming mice to induce mouse mastitis model,while the control group was injected with PBS.PBS,0.1%DMSO and indirubin were intraperitoneal injected at 1 h and 12 h to observe their effects on mice mastitis mice.Pathological changes and inflammatory cells were rarely seen in the control or DMSO groups.However,in the LPS group,mammary gland tissue had evident edema,inflammatory hyperemia,milk stasis,and local tissue necrosis;mammary alveoli were hyperemic,and thicker than in other controls,and neutrophil infiltration was seen in the alveolar lumen.Treatment with indirubin and Dex(Dexamethasone sodium phosphate injection)significantly ameliorated all LPS-induced macroscopic changes in a dose-dependent manner.Tissue in the LPS group had higher histological score than the control group(p < 0.001),and indirubin groups score reduced with the dose increasing.Indirubin inhibited the expression of TLR4 and played an important role in treatment of LPS-induced inflammation of MMECs and mouse mastitis.When treated with indirubin,TLR4 expression was sharply reduced with the increasing dose.Indirubin can suppress the activate of NF-?B signaling pathway induced by LPS,and played an important role in treatment of LPS-induced inflammation of MMECs and mouse mastitis.Indirubin can inhibit the m RNA expression of P50,P65 and I?B? and can induce the phosphorylation protein of P65 and I?B? by a dose dependent manner,played an important role in treatment of LPS-induced inflammation of MMECs and mouse mastitis.Indirubin can restrain the activation of mitogen-activated protein kinases(MAPKs)signaling pathway induced by LPS,take effect on LPS-induced inflammation within MMECs and LPS-induced mouse mastitis.Different concentrations of indirubin(25,50,and 100 n M)had no effect on non phosphorylated ERK,JNK and P38 protein level,but showed inhibition on the expression of phosphorylated P38,ERK and JNK induced by LPS.In the expression of m RNA,indirubin can inhibited the expression level of P38,ERK and JNK.Thus,we proved that indirubin can inhibit the activation of MAPKs pathway and play an important role in the treatment of LPS-induced inflammation of MMECs.4.Protective effect and mechanism of indirubin on LPS-induced mice mastitisAlthough the above results have been proved that indirubin have the effect on the treatment of mouse mastitis,nothing has been carried out to find the mechanism.Firstly,macroscopic pathology and histological analysis showed that indirubin can also significantly weaken LPS induced mouse breas.The comprehensive score showed that the score of LPS group was approximately 6 times higher than control group(p < 0.001),and the score of indirubin group decreased with the increasing dose of indirubin.ELISA was used to detect the expression of MPO,IL-1?,IL-6,and TNF-? in mammary tissue and serum from mastitis mice.Experimental results showed that indirubin gradually inhibited LPS-induced MPO,IL-1?,IL-6 and TNF-? with the increasing of its concentration,thus proved that indirubin had a largely potential role in defending LPS-induced mouse mastitis.However,whether the protective mechanism iscoincident with its therapeutic effects or not is still unclear.In the following study,we detected the protective mechanism of indirubin on LPS induced mouse mastitis in MMECs.ELISA and q RT-PCR results manifested that indirubin inhibited the expression of IL-1?,IL-6 and TNF-? in a dose dependent manner.Incubated 100 n M indirubin with MMECs for 24 h after LPS stimulation,indirubin started to inhibit three inflammatory factors at about 3h,reached at the lowest level at 9h,and stabilized after that.In COX-2 assay,indirubin inhibited COX-2 protein and m RNA expression rely on dose.Indirubin firstly inhibited COX-2 protein expression after inducted by LPS for 15 min,and stabilized at around 60 min.Then,indirubin inhibited the COX-2 RNA level at 3 hours,and stabilized nearly 9 hours.It can be concluded that indirubin can inhibit the expression of pro-inflammatory cytokines and the related inflammatory mediators,which can confront LPS induced inflammatory reaction in MMECs.We used q RT-PCR and western blot to determine whether indirubin inhibited the expression of TLR4.The result showed that indirubin can inhibited the expression of TLR4 at 25,50,and 100 n M,when concentration reached 100 n M,indirubin can effectively inhibit the protein and RNA expression of TLR4(p < 0.001).We used 100 n M indirubin pretreated MMECs for 24 h and induced by LPS,the results indicated that indirubin began to inhibit the expression of TLR4 protein at 15 min,inhibited the expression of RNA at 3h.Stabilized protein levels at the normal level after 60 min,and RNA level were relatively stable at around 9h.Since indirubin were involved in anti inflammation,NF-?B–associated signaling pathway was assessed by western blot.By inhibiting I?B? and P65 phosphorylation,indirubin challenged MMECs significantly suppressed NF-?B activity in LPS-stimulated cell in a dose-dependent manner.Protected by 100 n M indirubin,the phosphorylation were inhibited in LPS induced MMECs after 15 min,and stabilized after 60 min.MAPKs was assessed by western blot.Western blot results showed that indirubin challenge significantly suppressed MAPKs activity in LPS-stimulated MMECsby inhibiting P38,ERK and JNK phosphorylation in a dose-dependent manner.Indirubin inhibited MAPK at different time,it inhibited P38,ERK and JNK phosphorylation in15 min,30min and 45 min after induced by LPS,but the phosphorylation level was controlled at the normal range in about 60 min.5.The effects of indirubin to apoptosis on LPS-induced MMECsRhodamine phalloidin and DAPI were used to observe indirubin effects on morphological changes in MMECs.The results showed that indirubin improved LPS induced cases for MMECs,such as smaller deformation,turned round,cell membrane foamed,etc.apoptosis phenomenon in a dose dependent manner.LPS and indirubin induced MMECs had no effect on their sequence.Based on the study of apoptosis related genes,indirubin can activated anti apoptotic genes inhibited LPS stimulation,like Bcl-2,Bcl-x L,at both RNA and protein expression level.Indirubin can also inhibited the expression of pro-apoptosis genes,including Bax,Bak,caspase-3 and caspase-8 at m RNA and protein level.Therefore,indirubin had the function of inhibiting apoptosis the LPS stimulated inflammation in MMECs.In conclusion,our study firstly reported the function of indirubin in inhibiting TLR4,NF-?B,MAPKs and apoptosis related pathway related to mastitis induced by LPS.This study clarified the inhibition mechanism of chemokines IL-1?,IL-6,TNF-? and COX-2,demonstrated the pathogenesis mechanism of indirubin inhibited mastitis,and laid the theoretical foundation for treatment and prevention of bovine mastitis and other inflammatory disease.