| Endometritis which causes severe inflammatory response in the endometrial and muscularis of the uterus is one of the main diseases that leads to the decreased reproductive performance in female animals.Many factors can lead to endometritis,infection with pathogenic microorganisms,especially Escherichia coli(E.coli)and Staphylococcus aureus(S.aureus)which is most commonly.The invasion of pathogenic microorganisms causes an inflammatory response in the endometrial layer and myometrium,resulting in damage to the endometrium,which leads to reproductive disorders.Local or systemic administration of antibiotics is the common clinical treatment for endometritis.However,with the using of antibiotics is associated with many issues such as high levels of drug residues and significant toxic effects,especially the promotion of bacterial resistance.Therefore,it is necessary to find a new drug formulations to replace the antibiotics in the treatment of endometritis.。Chinese medicine has the characteristics of less resistance,low drug residue and less toxic side effects,among which Berberine(BBR)and Carvacrol(CAR),the active ingredients of plant origin,have antibacterial and anti-inflammatory pharmacological effects.The reason of why they have not been widely used in the treatment of veterinary clinical diseases was limited by the constraints of drug delivery method,formulation process and drug utilization rate.Temperature-sensitive chitosan(CS/β-GP)hydrogels have been used for the controlled release of various antibacterial drugs for the treatment of bacterial infectious diseases due to their adhesive,phase change and histocompatibility properties.Based on the pharmacological effects of BBR and CAR and the biological properties of CS/β-GP hydrogels,this study aims to prepare a CS/β-GP hydrogel loaded with BBR and CAR and conduct experimental clinical therapeutic trials.The research results may provide a scientific basis for the development of new traditional Chinese veterinary drugs for the treatment of endometritis,and provide new options for veterinary clinical drugs for the treatment of endometritis.The following three aspects of the study were tested:(1)In vitro screening of BBR and CAR for bacterial inhibition dosage,testing the inhibition effect of BBR and CAR(Minimum inhibition concentration(MIC),Minimum bactericidal concentration(MBC)and the grading between drugs.Evaluation of BBR and CAR alone or in combination for the inhibition of S.aureus and E.coli,together with screening of the amount of drug additive for subsequent hydrogel preparation.(2)Evaluation of berberine/carvacrol complex temperature-sensitive chitosan(BBR/CAR-CS/β-GP)hydrogels,CS/β-GP hydrogels and BBR/CAR-CS/β-GP hydrogels were successfully prepared;the characterization properties(gel time,p H,ultrastructure,rheological properties,drug slow release properties,swelling properties)and bacterial inhibitory effects of the gel systems were determined;the safety of BBR/CAR-CS/β-GP hydrogels on mouse uterus was evaluated.The mice were divided into a blank control group(CG)and a Chinese medicine composite gel group(CMG)to observe their mental status,leukocyte staining of secretions and uterine damage(uterine morphology,microstructure).These were aimed at screening the appropriate dose for the treatment of endometritis and evaluating the performance characterization and safety of the BBR/CAR-CS/β-GP hydrogel.(3)Study on the therapeutic effect of BBR/CAR-CS/β-GP hydrogel on endometritis in mice model.E.coli;and S.aureus endometritis models in mice were established.Mice were divided into blank control group(CG),model group(MG),herbal composite gel treatment group(CTG),blank gel treatment group(BTG),herbal composite gel group(CMG)and blank gel group(BG)to observe uterine damage(uterine morphology,microstructure),Leukocyte staining of uterine secretions,intrauterine antibacterial effect and inflammationrelated factors,aiming to evaluate the effects of BBR/CAR-CS/β-GP hydrogel on endometritis in mice.The experimental results were as follows:(1)Screening for in vitro bacterial inhibitory doses of BBR and CAR(1)The results of the anti-bacterial experiment showed that the MIC and MBC of BBR were 250 μg/m L and 500 μg/m L for both S.aureus and E.coli respectively,and the MIC and MBC of CAR were 125 μg/m L and 250 μg/m L for S.aureus,62.5 μg/m L and 1000 μg/m L for E.coli,respectively.(2)The FIC results showed that the combined BBR and CAR pairs were additive in the E.coli inhibition test and that neither the BBR nor CAR pairs were antagonistic in the S.aureus and E.coli inhibition tests.(3)The results of the combination showed that the drug combination of 1000 μg/m L CAR-and 500 μg/m L BBR had good antibacterial effect(2)Characterization performance,antibacterial performance and safety evaluation of BBR/CAR-CS/β-GP hydrogels.(1)CS/β-GP hydrogels and BBR/CAR-CS/β-GP hydrogels were successfully prepared.(2)The gelation time and p H results showed that the gelation time of CS/β-GP hydrogel and BBR/CAR-CS/β-GP hydrogel were 260 s and 280 s respectively,and the p H was 7.32 and7.4 respectively.(3)The scanning electron microscope results showed that the gels exhibited a threedimensional mesh structure,suggesting the gel system had good drug-carrying properties and mechanical elasticity.(4)The rheological results showed that the phase transition temperatures of CS/β-GP hydrogel and BBR/CAR-CS/β-GP hydrogel were 37.80℃ and 35.27℃ respectively,suggesting that the gel system could complete the gel phase transition with the in vivo temperature conditions.(5)The in vitro swelling results showed that the gel system had a swelling rate of 175.83%for CS/β-GP hydrogel and 166.00% for BBR/CAR-CS/β-GP hydrogel under physiological p H conditions,suggesting that the gel system had a good water absorption performance.(6)In vitro delayed release results showed that the cumulative drug release rate of the gel system could reach 60%～80% within 12 h,indicating that the gel system could delay the drug release rate.(7)The results of the in vitro anti-bacterial experiment inhibition showed that the BBR/CAR-CS/β-GP hydrogel significantly inhibited the growth of E.coli,S.aureus and their mixed bacteria.(8)The histopathological results showed that there was no significant damage to the morphology and microstructure of the uterus of CMG mice compared with CG,indicating that the BBR/CAR-CS/β-GP hydrogel had no significant stimulating effect on the uterus of mice.(9)The results of leukocyte staining of uterine secretions showed that only a few epithelial cells and scattered cell fragments were detected in the uterine secretions of CG mice,while the uterine secretion of CMG mice was similar to CG mice,with no obvious leukocyte aggregation.That indicated that BBR/CAR-CS/β-GP hydrogel did not induce an inflammatory response in the uterine cavity.(3)Study on the therapeutic effect of BBR/CAR-CS/β-GP hydrogel on mouse endometritis model(1)Histopathological results showed the uterine dissection of MG mice revealed bleeding points,and compared with CG mice,the microstructure showed inflammatory infiltration and pathological damage.Compared with MG,the inflammatory infiltration and pathological damage in the uterus of CTG mice were significantly alleviated,and the dissected uterus was light red in color with no obvious lesions.The above results indicated that the BBR/CARCS/β-GP hydrogel could effectively alleviate the uterine tissue damage caused by the mixed bacterial solution.(2)The results of leucocyte staining of uterine secretions showed that the number of MG mice leucocytes increased significantly compared with CG and Significantly lower CTG white blood cell count compared with MG mice,indicating that BBR/CAR-CS/β-GP hydrogel significantly reduced leucocyte aggregation in the inflammation-induced secretions.(3)The results of uterine cavity bacterial culture showed that the bacteria in uterine of MG mice was dominated by E.coli and S.aureus compared to CG mice,and CTG mice significantly reduced the number of pathogenic bacteria colonies.That indicated that BBR/CAR-CS/β-GP hydrogel significantly inhibited the growth of intrauterine pathogenic bacteria.(4)The results of inflammation-related factors assay showed that the expression levels of IL-6,IL-1β,TNF-α,IL-10,IFN-γ,TGF-β1,TLR4 genes and MPO activity levels were significantly increased in MG mice compared with CG(P<0.05,P<0.01).The expression levels of the above gene and MPO activity levels were significantly lower in CTG mice compared with MG(P<0.05,P<0.01).The above results indicated that the BBR/CAR-CS/β-GP hydrogel could alleviate the inflammation caused by the mixed bacterial solution.In conclusion,this study successfully prepared BBR/CAR-CS/β-GP temperaturesensitive hydrogel,which has good carrier properties and antibacterial activity,and has good therapeutic effect on endometritis in mice,providing a new choice of therapeutic drug for veterinary clinical endometritis. |
PDF Full Text Request