Anti-inflammatory Effects Of Hederacoside-C Via Suppressing TLR2&TLR4 And Their Downstream NF-κB And MAPKS Signaling Cascade

In current era,the dairy cows are experiencing tremendous stress and severely increasing their susceptibility to the metabolic,infectious and inflammatory diseases.Mastitis continues to be the most familiar inflammatory disease afflicting dairy cows during high yield period.During mastitis,milk production is reduced,quality is impaired and veterinary cost is increased.Total annual economic losses attributable to mastitis had been estimated approximately $1.8 billion in US and ￥15–45 billion in China.Among all etiological lethal agents,predominantly,Staphylococcus aureus(S.aureus)is the most common and contagious pathogen causing life-threatening complications in humans and animals.Ruminant is one of the most important reservoir of pathogenic S.aureus and cow is an important ruminant which serves as an essential food animal for human.Cow is also susceptible to S.aureus mastitis and some other infectious diseases.S.aureus-associated mastitis in cows can only be treated effectively by administering “Antibiotics”.However,due to widespread consumption of antibiotics in veterinary fields,S.aureus pathogenically and virulently has widely emerged to antibiotic resistance causing serious food safety and public health problems,and antibiotics were now prohibited in dairy industry in many countries.In the given scenario,to facilitate the prevention and efficient treatment of S.aureus-induced mastitis,there is an urgent need of an effective strategy particularly when herd management is failed.Herbal medicines i.e.Tradition Chinese Medicines(TCM)have promising therapeutic effects against bacterialassociated inflammatory diseases,indicating its broad prospect of application in food animal industry.The objective of this project was to elucidate the S.aureus-induced TLRs-mediated underlying pathways followed by the expressions of pro-inflammatory and antiinflammatory cytokines,and therefore provides scientific basis to the use of TCM(Hedera helix extract;HDC)for the treatment of mastitis.1.Clinical Histopathology and Inflammatory Factors in Mastitis of Dairy CowsTo the best of our knowledge,mastitis is the inflammation of mammary glands,and inflammation or any tissue injury activates Toll-like Receptors(TLRs).Activated TLRsmediated NF-κB/MAPKs pathways initiate the gene expression of pro-inflammatory cytokines followed by innate immune response and then cause patho-physiological changes.Cow mammary gland tissues were collected from Local Abattoir of Wuhan,Hubei,China,and were set for histopathological investigation and analysis of various inflammatory mediators.The results indicated severe histopathological changes in infected mammary glands with abundant poly-morphonuclear cells,gland alveolar lumen with compromised integrity,edema and cellular debris.Milk ducts and secretory acini contained cellular exudate with profuse infiltration of mononuclear cells intermixed with neutrophils were also observed.Moreover,isolates of S.aureus from infected mammary gland tissues were successfully identified on Sheep Blood Agar and Mannitol Salt Agar exhibiting beta haemolysis and white to yellowish colour colonies respectively.S.aureus also exhibited positively with catalase,coagulase and mannitol fermentation tests.Western blot and q RT-PCR results suggested the significant(P < 0.05)expressions of TLR2/TLR4 associated with NF-κB/MAPKs pathways and significant(P < 0.05)upregulation of pro-inflammatory cytokines(TNF-α,IL-1β and IL-6)in infected tissues rather than non-infected tissues.These results suggested that cow mastitis was commonly caused by S.aureus which severely damaged the mammary gland tissues,and hence the risk of invasive bacterial disease “mastitis” in cows was significantly high.Due to emerging antibiotic resistance and public health problems,many countries have limited the use of antibiotics to treat bacterial infections in animal industry.A large number of clinical practices revealed that the toxicity and drug resistance of Traditional Chinese Medicine were negligible.Therefore,in this thesis,the anti-inflammatory drug Hederacoside-C(HDC)was used to investigate its role in inhibiting the inflammatory response and damages in mastitis and to elucidate its mechanism of actions.2.Hederacoside-C Inhibiting Inflammatory Response via TLR2 & TLR4 and Their Downstream Signalling NF-κB & MAPKs Pathways in-vitroThis study aimed to evaluate the anti-inflammatory effects of HDC on an in-vitro mastitis model against major mastitis causing pathogen,S.aureus and to elucidate the potential mechanisms responsible for anti-inflammatory effects of HDC on S.aureusinduced RAW 264.7 cells inflammation.The cytotoxicity on RAW 264.7 cells was estimated with CCK-8 assay and these results suggested that HDC had no toxic effects on RAW 264.7 cells.Further,ELISA and q RT-PCR results indicated that HDC with increasing concentrations,significantly(P < 0.05)suppressed the production of proinflammatory cytokines TNF-α,IL-1β and IL-6 by downregulating their relevant genes and significantly(P < 0.05)promoted the release of anti-inflammatory cytokine IL-10 by upregulating its relevant genes.Furthermore,TLRs,NF-κB and MAPKs pathways were investigated with western blot analysis.Molecular research on TLRs revealed that HDC evidently(P < 0.05)inhibited the activation of S.aureus-stimulated TLR2 and TLR4.Moreover,our results also demonstrated that HDC significantly(P < 0.05)suppressed the phosphorylation of S.aureus-triggered p65,IκBα,p38,ERK and JNK proteins in dose dependent manner.The above results suggested that HDC effectively(P < 0.05)blocked the TLRs-mediated NF-κB and MAPKs signaling cascade triggered by S.aureus in RAW 264.7 cells by effectively(P < 0.05)blocking the inflammatory response and inflammatory damages of the cells.Thus,HDC might be a potential effective Chinese medicine against S.aureus-induced inflammation via exerting protective antiinflammatory effects on RAW 264.7 cells inflammation.3.Hederacoside-C Suppressing Inflammatory Response in Mastitis via Inhibiting TLR2 & TLR4 and Their Downstream Signalling NF-κB & MAPKs Pathways invivoTo investigate the anti-inflammatory effects of HDC on S.aureus-induced mastitis,we designed BALB/c mice mastitis model using different concentrations of HDC,5 mg/Kg,10 mg/Kg and 50 mg/Kg in-vivo and evaluated the changes of inflammatory mediators and gene expression levels in mammary gland tissues.The ascertained histopathological changes and MPO activity revealed that HDC significantly(P < 0.05)defended mammary glands from tissue destruction and inflammatory cell infiltration induced by S.aureus.The results of ELISA,western blot and q RT-PCR indicated that HDC markedly(P < 0.05)suppressed the TLR2 & TLR4 expressions,significantly(P < 0.05)decreased the phosphorylation of p38,ERK,JNK,p65 and IκBα,and thereby significantly(P < 0.05)inhibited the expressions of TNF-α,IL-1β & IL-6 and enhanced the IL-10.Conclusion: In RAW 264.7 cells mastitis model,we determined the molecular mechanisms related to drug anti-inflammatory property and in mice mastitis model we confirmed inflammatory mechanisms and drug effects.Summarizing the whole study,HDC significantly(P < 0.05)suppressed the TLR2 & TLR4 expressions and then attenuated the MAPKs(p38,ERK,JNK)and NF-κB(p65 & IκBα)pathways followed by decreasing the phosphorylation of p38,ERK,JNK,p65 and IκBα significantly(P < 0.05).This effect and mechanism of HDC enhanced the mammary gland defence and reduced inflammatory injuries.These findings suggested that HDC might have been a potential effective anti-inflammatory drug for the S.aureus-induced mice mastitis.Findings:(1)S.aureus is one of the pathogenic bacteria involved in mastitis.(2)HDC had greatest potential value of anti-inflammation against S.aureus to mastitis.(3)Its molecular mechanism might be: HDC attenuated the MAPKs and NF-κB pathways by suppressing the TLR2 & TLR4 expressions,and then inhibited TNF-α,IL-1β & IL-6 expressions and enhanced the IL-10 expression.