Changes Of Energy Metabolism And Immune Status In Peripartal Cows And Their Regulation Of Ligustrum Lucidum

Peripartal cows often experience negative energy balance(NEB)and are prone to metabolic and infectious diseases,which affects the economic development of dairy industry.Sound and sensitive population monitoring technology can guarantee the health and production of dairy cows.Clarifying metabolic and immune characteristics of peripartal cows can provide data basis for early warning and prevention of peripartal metabolic and infectious diseases.At present,the studies on the immune function of peripartal cows mainly focus on the concentration of cytokines in vivo and the changes in the activity and function of some cells in vitro,and lack systematic evaluation on the immune changes of cows and the changes in the function of neutrophils in vitro.Ligustrum lucidum is a kind of traditional Chinese medicine with various effects,which can improve the performance of livestock and poultry production and has protective regulation effects on lipid metabolism and immune function of livestock and poultry.However,Ligustrum lucidum is rarely used in dairy cow production,and its beneficial effects on metabolic and immune disorders of peripartal cows remains unclear.Therefore,the present study first examined the energy metabolic status and immune status(changes in cytokine levels,immune cell composition,and neutrophil function)of cows from 5 w before delivery to 3 w after delivery.Related results of energy metabolism showed that the serum concentration of nonesterified fatty acids(NEFA)and beta-hydroxybutyric acid(BHBA)in dairy cows increased after delivery,while the serum glucose concentration decreased after increasing on the day of delivery,indicating that cows experienced physiological NEB after delivery.Cytokine results showed that the concentration of interleukin(IL)-1βdecreased after delivery,the concentration of IL-2 increased from 1 w before delivery to 3 w after delivery,and the concentration of IL-6 and IL-8 decreased before and after delivery,indicating that the cytokine levels of dairy cows fluctuated significantly during peripartal period.The results of immune cell composition showed that the proportion of neutrophils increased and the proportion of lymphocytes decreased before and after delivery.Among T cells,the proportion of CD4~+T cells decreased only on the day of delivery,while that of CD8~+T cells decreased after delivery,and the ratio of CD4~+T cells to CD8~+T cells increased in the peripartal period,except for a sharp decrease on the day of delivery.These results indicated that the proportion of immune cells fluctuated significantly in the peripartal period.In vitro tests of neutrophil function showed that phagocytosis function was enhanced from 2 w before delivery,reactive oxygen species release level increased throughout the peripartal period(3 w before delivery to 3 w after delivery),and neutrophil extracellular traps(NETs)release level decreased from 1 w before delivery to 3 w after delivery,indicating that the function of neutrophils in dairy cows varied dramatically in the peripartal period.The decreased NETs release might be one of the reasons for the high incidence of infectious diseases in peripartal cows.Subsequently,we tested the regulatory effects of Ligustrum lucidum on energy metabolism and immune function of peripartal cows.The results of energy metabolism showed that Ligustrum lucidum could significantly reduce the serum concentration of NEFA and BHBA in postpartum dairy cows,indicating that Ligustrum lucidum had a regulatory effect on physiological NEB of dairy cows.The results of cytokines showed that Ligustrum lucidum could attenuate the increase of IL-2 and the decrease of IL-8 in postpartum cow,suggesting that Ligustrum lucidum could improve postpartum cytokine homeostasis.The results of immune cell composition showed that Ligustrum lucidum could relieve the increase of neutrophil proportion and the decrease of lymphocyte proportion before and after delivery,indicating that Ligustrum lucidum could improve the balance of immune cells in peripartal cows.Ligustrum lucidum could correct the decrease of CD4~+T cell ratio on the day of delivery and the decrease of CD8~+T cell ratio after delivery,and promote the stability of CD4~+T cell/CD8~+T cell ratio,indicating that Ligustrum lucidum had a positive effect on the balance of T cell immunity in cows.The results of neutrophil function showed that Ligustrum lucidum could improve the function of neutrophil phagocytosis and NETs release in peripartal cows.Then we treated neutrophils with oleanolic acid(OA)and salidroside respectively in vitro to detect the effects of OA and salidroside on the function of neutrophils.The results showed that OA could enhance NETs release,and salidroside could enhance neutrophil phagocytosis and NETs release,suggesting that OA and salidroside might be one of the reasons why Ligustrum lucidum played a role in dairy cows.Finally,we investigated the impaired NETs release in peripartal cows and the underlying mechanism.High concentration of NEFA in the blood of peripartal cows is lipotoxic and can affect the function of immune cells.Palmitic acid(PA)is the most abundant saturated fatty acid in the blood,which may be the pathogenic factor for the damaged NETs release in peripartal cows.Therefore,we treated peripartal cow neutrophils with PA in vitro to explore the effect of PA on NETs release and its potential mechanism.The results showed that PA could reduce the level of double strain deoxyribonucleic acid(ds DNA)released by neutrophils,the expression of cit-histone and the proportion of cells releasing NETs,indicating PA inhibited NETs release.Autophagy is a key process in the regulation of NETs.The results showed that PA could induce the protein accumulation of p62 and LC3-II,indicating that PA inhibited neutrophil autophagy in dairy cows.In addition,pretreatment with rapamycin,an autophagy activator,weakened the inhibitory effect of PA on the release of NETs in dairy cows,suggesting that PA reduced the release of NETs in dairy cows by inhibiting autophagy,which might be a reason for the immune dysfunction in dairy cows during the peripartal period.In conclusion,the immune status of cows fluctuated during the peripartal period(IL-1β,IL-6,IL-8 levels decreased,IL-2 levels increased;the proportion of neutrophils increased and the proportion of lymphocytes decreased;the release of NETs was weakened and neutrophil phagocytosis were enhanced).Ligustrum Ligustrum could promote energy metabolism(reducing the levels of NEFA and BHBA)and immune status(relieving the increase of IL-2 and decrease of IL-8;relieving he increase of neutrophil proportion and the decrease of lymphocyte proportion)to return to homeostasis in peripartal dairy cows.Ligustrum Ligustrum and its active monomers OA and salidroside could enhance the release of NETs.PA could reduce the release of NETs by inhibiting autophagy.