| Dairy cow laminitis is the diffuse,slurry,and sterile inflammation of the dairy cow hoof claw.This disease has been considered as the third most serious disease,behind mastitis and reproductive problems in dairy cows in China.Therefore,based on the successful use of oligofructose to induce acute laminitis in dairy cows,this study explored the roles of oxidative stress,apoptosis,and autophagy in the occurrence and development of the disease at the gene,protein,and tissue levels,respectively,and observed the microscopic and ultrastructural damage of laminar tissue,aiming to provide a theoretical basis for the pathogenesis study and the development of new diagnosis and treatment techniques for laminitis in dairy cows.Twelve healthy non-pregnant Chinese Holstein cows were randomly divided into 2 groups with 6 cows in each group,which were named control group and oligofructose overload group(OF group),respectively.At 0 h,cows in OF group were gavaged 17 g/kg of oligofructose solution through oral rumen tube,and dairy cows in control group were gavaged with the same volume of deionized water by the same method.At-72 h before perfusion,0,6,12,18,24,36,48,60,and 72 h,clinical signs in control group and OF group were monitored,and blood from the jugular vein was collected.After 72 h,laminar tissues of dairy cows in the control group and the OF group were collected.And collected laminar tissues from cows were prepared into tissue slices.By characteristic histopathological changes confirmed that acute laminitis model of dairy cows was successfully established.In this study,the changes of oxidative stress related indexes in serum samples of dairy cows were detected;ultrastructural damage was observed by t ransmission electron microscopy,and gene expressions of oxidative stress,apoptosis,and autophagy-related indexes were detected by real-time fluorescence quantitative technique;the protein expressions of oxidative stress,apoptosis,and autophagy-related indexes were detected by western blotting;the number and distribution of specific cells associated with oxidative stress,apoptosis,and autophagy were observed by immunohistochemical techniques.The experimental results showed:(1)Compared with the control group,the dairy cows in the OF group showed depression,loss of appetite,watery diarrhea,weight shift,obvious swelling of coronet band,hyperpulsatile toe artery,continuous pain of hoof claws,obvious lameness,increased heart rate,decreased respiratory rate,increased rectal temperature,increased hoof capsule temperature,slowed down rumen contraction speed,and decreased p H value of rumen fluid.Control cows showed no signs of systemic disease.(2)Histopathological observation of dairy cow lamellae in the OF group showed that the lamellae layer was elongated,the dermal lamellae was edema and hemorrhage,the cell layer was increased,the basement membrane was blurred and wavy,the cubic basal cells had circular light-stained nuclei,and the infiltration of monocytes and granulocytes was common.In the control group,the laminar tissue had clear basement membrane,columnar basal cells with dark nuclei,and very little infiltration of monocytes and granulocytes.The results of the dairy cow lamellae in the OF group showed the characteristic pathological changes of acute laminitis.(3)In ultrastructural observation of lamellae from dairy cows with laminitis,the tip of basal cells of epidermis became sharp;the dense layer was broken,and the lightly stained collagen fibers were connected with each other;the number of hemidesmosomes on the cell membrane decreased;the distance between the basal cells and the epidermal lamellae incr eased;epidermal basal cells have deformed nuclei abnormally close to the basement membrane region with blurred boundaries and reduced chromatin than in control cows.(4)Compared with the control group,the average activity of superoxide dismutase(SOD)i n serum of dairy cows with laminitis was significantly decreased a t 18 h(P<0.05),and extremely significantly decreased at 24 h to 72 h(P<0.01).Compared with the control group,the serum malondialdehyde(MDA)concentration of cows in the OF group was significantly increased from12 h to 18 h(P<0.05)and was extremely significantly increased from 24 h to 72 h(P< 0.01).There were no statistically significant differences in the activities of GSH and CAT in the serum of OF dairy cows compared to the control group.(5)Gene expression results of oxidative stress,apoptosis,and autophagy-related indicators in the lamellae of dairy cows with laminitis: compared with the control group,the m RNA expression levels of Keap1,Bax,and Beclin1 in the lamellae of cows with laminitis were significantly increased(P<0.01).The m RNA expression levels of P53,ATG5,and ATG12 were significantly increased(P<0.05),while the m RNA expression levels of Nrf2,Ho1,Bcl2,P62,and m TOR were extremely significantly decreased(P<0.01),and the m RNA expression level of Nqo1 was significantly decreased(P<0.05).(6)The protein expression results of oxidative stress,apoptosis,and autophagy-related indicators in the lamellae of dairy cows with laminitis: compared with the control group,the protein expression levels of Keap1,Bax,Bif1,and Caspase3 in the lamellae of dairy cows with laminitis were extremely significantly increased(P<0.01).The protein expression levels of Caspase8,Caspase9/9p,and Beclin1 were significantly increased(P<0.05),while the protein expression levels of Nrf2,Nqo1,and Bcl2 were extremely significantly decreased(P<0.01),and the protein expression level of Ho1,and LC3 b were significantly decreased(P<0.05).(7)Compared with the control group,the distribution of Bax,P53 and Beclin1 proteins were extremely significantly increased(P<0.01),and the distribution of Keap1 protein was significantly increased(P<0.05).However,the distribution of Nrf2 and Ho1 proteins were extremely significantly decreased(P<0.01),and the distribution of P62 protein was significantly decreased(P<0.05).In conclusion:(1)The model of acute laminitis in dairy cows was successfully established by the method of oligofructose overload,and the dairy cows in the OF group showed the characteristic clinical symptoms and histopathological changes of acute laminitis.(2)The dense layer of the lamellae of the dairy cows with laminitis was damaged,the collagen fibers were stained light,the number of hemidesmosomes was reduced,and the basal cell nuclei were deformed,which proved the ultrastructural damage of the laminar structural cells during the onset of laminitis and provided the basis for the histopathological changes of the lamellae of the dairy cows.(3)The detection of oxidative stress-related indicators showed that the blood SOD activity decreased and MDA concentration increased in dairy cows with laminitis,indicating that the oxidation-reduction balance was destroyed during the onset of laminitis and systemic oxidat ive stress injury occurred.(4)The results of gene expression,protein expression,and immunohistochemistry of oxidative stress,apoptosis,and autophagy-related indicators in the lamellae of dairy cows with laminitis indicated that oxidative stress injury occurred during the onset of laminitis,which promoted the occurrence of apoptosis,and autophagy,and confirmed that oxidative stress,apoptosis and autophagy were involved in the pathogenesis and regulation of laminitis in dairy cows. |
PDF Full Text Request