| In recent years,the amount of fattening sheep increase year by year,and the economic benefits are better and better.But in the feeding process of sheep,follow with some diseases.In order to improve the growth rate of fattening sheep,add much more high-energy feed,which often leads to the occurrence of urinary calculus.Urinary calculi is one of the common diseases of fattening sheep,it can cause inflammation of urethra and bladder.In severe cases,it can lead to urethral obstruction,which can lead to the death of animals due to secondary uremia,this seriously restricts the development of sheep industry.As a metabolic disease,the key to sheep urinary calculi is prevention.But so far,no effective prevention and control measures have been found.In this study,through the epidemiological investigation of sheep urinary calculi in Baoding area,to reveal the regularity of the disease.By the establishment of sheep renal tubular epithelial cells(RTECs)injury model,the protective effects of poria cocos polysaccharide(PCP)and total flavonoids of Lysimachia christinae on sheep RTECs injury were explored,so as to provide a basis for the prevention and treatment of sheep urinary calculi with traditional Chinese medicine.The epidemiology of sheep urinary calculi in Baoding area was investigated,and the relationships of sheep urinary calculi formation with diet,water-drinking amount,age,season and additives etc.It found that among the calculi cases that occurred,phosphate calculus was the main type in this area,part of calculi were calcium-type.Bladder calculi and urethral calculi are the most common calculi,their incidence rates were 45.91%and 38.34%respectively.The occurrence of sheep urinary calculi was closely related to age,season,gender,water-drinking amount and diet.In terms of age,the incidence rate of sheep aged 2～5 months showed an upward trend,and the incidence rate of sheep at the age of 5 months was the highest,up to 2.97%.The incidence rate of sheep urinary calculi in winter was much higher than other seasons,which was 3.83%.The incidence rate of rams was much higher than that of ewes,up to 10.86%.The incidence of sheep urinary calculi who drank water four times a day on average and added warm water twice in winter was low,which was 6.31%.The study also showed that the incidence rate of urinary calculi in sheep farms that used anti-calculi additives were lower than those of sheep farms without taking measures.The incidence rate of urinary calculi in sheep farms that used ammonium chloride as a preventive measures was the lowest,accounting for 6.42%.The incidence rate of urinary calculi in sheep farms without taking any preventive measures was 10.42%,which was the highest.The injury model was established by stimulating sheep RTECs with magnesium ammonium phosphate(MAP),and the injury effect of MAP on RTECs was explored.The results showed that sheep RTECs with good activity and high purity were isolated and cultured successfully by collagenase digestion.10 mg/mL MAP was the optimal concentration for RTECs injury by CCK-8 method determination.By western blot,ELISA and qPCR methods,it showed that MAP could significantly reduce the activities of sheep RTECs and promote the secretions of IL-6 and TNF-α.As the concentration of MAP increased,the expression of MDA and the activity of LDH increased gradually,while the activity of SOD decreased.The expressions of OPN,VCAM-1 and ICAM-1 increased by MAP concentration dependent manner.In MAP group,the phosphorylation of sheep RTECs IκBα and NF-κB p65 proteins increased,while the expressions of Nrf2,NQO1 and HO-1 were decreased.The expression of apoptotic protein caspase-3 increased significantly,and the ratio of Bcl-2/Bax decreased.By sheep RTECs damage model,the protective effects of PCP and total flavonoids of Lysimachia christinae on MAP-induced sheep RTECs injury were investigated.The results showed that both drugs could reduce the oxidative stress of sheep RTECs induced by MAP,which was manifested by a decrease in the levels of lactate dehydrogenase(LDH)and malondialdehyde(MDA),and an increase in superoxide dismutase(SOD)activity.It could also inhibit the induction of inflammatory cytokines IL-6 and TNF-α by MAP.After drug treatment,it showed that the protein expression levels of Nrf2,NQO1 and HO-1 were significantly higher than those in MAP group,and the relative expression levels of p-NF-κB p65 and p-IκBα were significantly lower than those in MAP group.By qPCR,it showed that the expressions of OPN,VCAM-1 and ICAM-1 mRNA decreased with the increase of drug concentration.The preventive and therapeutic effects of PCP and total flavonoids of Lysimachia christinae on sheep urinary calculi were explored through the detection of relevant biochemical indices.The results showed that adding PCP and total flavonoids of Lysimachia christinis in the diet could effectively reduce the incidence rate of sheep urinary calculi.In prevention group 2,the quantity of urinary sediments reduced,and the levels of sera creatinine and urea nitrogen also decreased significantly.The magnesium ions and phosphorus ions in blood and urine were significantly lower than those in prevention group 1 and treatment group.In comparison,the preventive effect of the group 2 is the best,and the combination of PCP and total flavonoids of Lysimachia christinae has a certain therapeutic effect on sheep urinary calculi.In conclusion,in this study the incidence regularity and epidemic characteristics of sheep urinary calculi in Baoding area was discussed.Sheep RTECs were successfully isolated and cultured,and the cell damage model was established,and the mechanism of MAP on damage to sheep RTECs was preliminarily explored.PCP and total flavonoids of Lysimachia christinae can protect sheep RTECs from oxidative stress and inflammation induced by MAP,and reduce the incidence of sheep urinary calculi,which can provide reference for the prevention and treatment of sheep urinary calculi in clinic. |
