Protective Effect Of Osthole On LPS-induced Mastitis In Mice

Dairy cow mastitis is the inflammation of mammary gland because of various stimulation including physical,chemical,microbiological and other pathogenic factors.It can cause serious problems such as reducing lactation and milk quality,and huge economic losses to the breeding industry.Osthole,the main active ingredient of Cnidium,has various biological activities including anti-inflammatory,anti-bacterial and anti-oxidant,but it has not been reported in the prevention and treatment of mastitis.In this study,the safety of osthole was evaluated d by acute toxicity test,reevaluated the prevention and treatment effect of osthole on LPS-induced mastitis in mice were tested by bacteriostatic test in vitro and animal experiments in vivo.The study results may provide reference for the drugdevelopment in the prevention and treatment of mastitis.（1）Acute toxicity test The LD₀ and LD₁₀₀ of osthole were determined by the method of intraperitoneal injection in the preliminary test,and the poisoning performance of mice and calculate LD₅₀ were assayed in the formal test.（2）In vitro antibacterial test Agar plate diffusion method was used to determine the antibacterial activity of osthole against Staphylococcus aureus,Escherichia coli and Streptococcus agalactiae.（3）Protective test of osthole on LPS-induced mouse mastitis The mice were divided into control group,model group,osthole low,medium and high dose groups（20,30 and 40 mg/kg）,dexamethasone（Dex）group,LPS solvent control group and osthole solvent control group.Mice mastitis model was established by the breast ducts injection of LPS.Mice were intraperitoneally injected with different doses of osthole at 1 h before and 12 h after injection of LPS.At the end of the experiment,the pathological,anatomical and histological changes of breast tissues were observed;the activity of MPO,the levels of pro-inflammatory factors IL-1β,IL-6 and TNF-α,and the levels of oxidative stress indexes NO,T-SOD,MDA,GSH-PX and CAT were detected by the kit.The results showed that the LD₅₀ of osthole was 464.62 mg/kg,and the 95%confidence limit was 398.66-541.25 mg/kg.According to pharmacological toxicological classification,osthole belonged to a low toxic substance.More than or equal to 1.5mg/m Lof osthole’concentration had certain inhibitory effect on the three pathogenic bacterias,and the diameter of the inhibition zone was more than 14 mm.In the protective test of osthole on LPS-induced mastitis in mice,the mammary gland tissues of the model group were obviously red and congested.The histological observation showed that the acinar wall of the mammary gland was significantly thickened,and the acinar cavity was infiltrated by a large number of inflammatory cells,and the structure of the vesicles was destroyed.The breast tissues of the control group had no obvious pathological changes with the macroscope and histology examination of.The pathological damage of the breast tissues in the three osthole groups were improved to some extent,and the pathological changes were relatively slight in 30 mg/kg and 40 mg/kg dose groups.The test results of the kit showed that compared with the model group,the 3 doses of osthole significantly reduced NO and MDA content,MPO activity,IL-1β,IL-6 and TNF-αlevels,and increased T-SOD,GSH-PX and CAT content（P<0.01）.In conclusion,the dosages of 20,30 and 40 mg/kg of osthole had a protective effect on LPS-induced mastitis in mice,especially the doses of 30 mg/kg and 40 mg/kg.