| Chronic stress(CS)refers to the stress that the stimulus lasts for a long time and causes a longer duration of the body response.Long-term feeding in restricted pens will cause CS in pregnant sows,resulting in decreased immunity of sows and increased disease incidence,which will affect the long-term benefits of the pig industry.As the pathogenesis of decrease in immunity caused by CS is unknown,it is important to explore the pathogenic mechanism of CS and the effective therapeutic drugs.Studies have shown that CS can cause an unfolded protein reaction in the endoplasmic reticulum of the spleen,mitochondria and endoplasmic reticulum are two organelles which can determine the fate of cells.In the case of pathology,oxidative stress happens and the two organelles are closed to each other to form Mitochondria-associated Endoplasmic Reticulum membranes(MAMs)and quickly build IP3R/GRP75/VDAC1 calcium ion fast channels in this area to connect the two organelles closely in structure and function to respond to external stimuli.However,whether this pathological mechanism is involved in the mechanism of CS-induced spleen injury is not yet clear.Melatonin(MT)can directly act on the outer mitochondrial membrane,reduce ROS production,and play an important role in antioxidant,immune and HPA axis regulation.Therefore,it is speculated that MT can reduce mitochondrial ROS production,weaken the unfolding of the endoplasmic reticulum protein response,decrease MAMs production and adjust the mitochondrial dynamics imbalance to play a protective role in CS-induced spleen damage.Then through the pig farm regression test,explore the effect of MT in alleviating the CS of pregnant sows caused by feeding in restricted pens.In preliminary pig farm experiment,we conducted behavioral observation,entered the information of pregnant sows,and then collected blood sampling from ear veins to detect blood routine indicators,HPA axis-related indicators,oxidative stress-related indicators,and immunity related indicators.In order to explore the impact of CS on the immune system and its solutions,in the laboratory stage,we divided SD pregnant rats into four groups,C group,CS group,CS+M group(10 mg/kg MT)and CS+A group.Rats were restrained for 18 days for 6 hours a day to establish the rat CS model,and injected intraperitoneally 1 hour before restraint every day.On the19 th day,6 pregnant mice were selected from each group for an open field experiment,performed blood collection and sampling and detected spleen structure,HPA axis-related indicators,oxidative stress-related indicators,immune-related indicators,m RNA and protein expression level of mitochondrial dynamics,endoplasmic reticulum stress and mitochondrial-endoplasmic reticulum structure coupling(MAMs).In order to verify the therapeutic effect of MT on the immune decline caused by CS,we carried out a pig farm regression test.According to the statistical results of the previous pig farm experiment,we took the restricted feeding of the fifth-parity pregnant sows as the research object.On the 21 st day of the restriction(the 50 th day of pregnancy),MT was added in the morning at a dose of 3 mg/day and fed for 21 days.Before feeding on the 71 st day of pregnancy,blood was collected from the ear veins to detect blood routine indicators,HPA axis-related indicators,oxidative stress-related indicators,and immune-related indicators.Compare the therapeutic effect of MT on the immunological decline caused by CS in pregnant sows.The results are as follows:(1)Pregnancy sows in restricted feeding have reduced feed intake,leftover feed in the food bowl,sluggishness,slow response,loss of interest in surrounding things,and often show habits of nibbling pens,troughs and chewing without food.And among the related indicators of the HPA axis,the levels of CRF,ACTH and CORT increased significantly,DA and 5-HT decreased significantly,and the HPA axis was activated,indicating that restricted feeding led to the occurrence of CS in pregnant sows.(2)After restricted feeding causes CS in pregnant sows,serum GSH,GSH-Px content,CAT,SOD,and GSH-Px activities are significantly reduced,and MDA content is significantly increased,indicating that restricted feeding can cause pregnant sows.The body’s oxidative capacity is enhanced,and antioxidant capacity is weakened,leading to oxidative stress.The number of white blood cells and lymphocytes,the percentage of lymphocytes in the plasma decrease,the percentage of neutrophils increases,the content of CD3 and CD4 decrease significantly,the CD8 increase,and the percentage of CD4/CD8 decrease indicated that restricted feeding can cause the immunity of pregnant pigs decrease.(3)In the open field experiment of chronic stress rats,the resting time was significantly shortened,the total distance of exercise,the number of straddle times and the number of standing times were significantly increased,the levels of CORT,CRF and ACTH in the serum were significantly decreased,and the levels of DA and 5-HT were significantly increased,indicating that the rat’s desire to explore is reduced,the HPA axis is activated,and the CS model is successfully established.(4)The contents of GSH and GSH-Px in the spleen of chronic stress rats were significantly reduced,the activities of CAT and SOD were significantly reduced,and the contents of MDA and ROS were significantly increased.After MT administration,the content of GSH increased significantly,the activities of CAT,SOD,and GSH-Px increased significantly,and the content of MDA and ROS decreased significantly.It shows that CS can cause oxidative stress in the spleen of rats,and MT plays a protective role.The white blood cells and lymphocytes in the blood of CS rats were significantly reduced,the percentage of lymphocytes was significantly reduced,the percentage of neutrophils was significantly increased,the content of CD3 and CD4 was significantly reduced,the content of CD8 was significantly increased,and the percentage of CD4/CD8 was significantly reduced.After MT administration,the abnormal index was reversed.It shows that MT can alleviate the decrease in immunity of rats caused by CS.(5)The spleen of CS rats was significantly enlarged,and the spleen coefficient was significantly increased,histopathological observations showed that the structure of the spleen was damaged,the white pulp structure was loose and disordered,the central artery was swollen,and the marginal area partially disappeared,and the white pulp and red pulp were unclear.The results of electron microscopy showed that the ultrastructure of spleen cells was damaged,the nucleus was pyknotic and densely stained,the endoplasmic reticulum and mitochondria swelled increased in number,and the mitochondrial-endoplasmic reticulum structure coupling increased.After MT administration,the morphology and structure returned to normal.It shows that MT can protect the spleen structure of CS rats.(6)The m RNA level of Drp1 and Fis1 in the spleen cells of CS rats increased significantly,the m RNA level of Opa1,Mfn1,and Mfn2 decreased significantly,the ratio of p-Drp1 S616/Drp1 increased significantly,and the protein expression level of Fis1 increased significantly,the protein expression level of Opa1,Mfn1 and Mfn2 was significantly reduced,and MT administration reversed the abnormality of the indicators.It shows that CS can cause the imbalance of mitochondrial dynamics in the spleen of rats,and MT can relieve it.The m RNA level of IRE1,ATF6,and PERK in the spleen cells of CS rats increased significantly,and the m RNA level of GRP78 did not change significantly,the protein expression level of p-IRE1 a,ATF6a and p-EIF2 a increased significantly,and the protein expression level of GRP78 increased significantly.There was no significant change in protein expression.After MT administration,the abnormal index was reversed and returned to the level of the control group,indicating that CS can cause endoplasmic reticulum stress in rat spleen cells,and MT can relieve it.The m RNA and protein expressions level of IP3 R,GRP75,and VDAC1 in the spleen cells of CS rats were significantly increased.After MT administration,the abnormal indicators were reversed,indicating that CS can cause mitochondrial-endoplasmic reticulum coupling in rat spleen cells,MT plays a mitigating effect on it.(7)The level of serum CORT,CRF and ACTH increased significantly,and the levels of DA and 5-HT decreased significantly.MT administration reversed the abnormality of HPA axis related indicators,indicating that MT plays a protective role in CS of pregnancy sows caused by restricted feeding.(8)The content of serum GSH,CAT and GSH-Px activity of sows in restricted feeding gestation were significantly reduced,the content of SOD was reduced,but there was no significant difference,and the content of MDA was significantly increased.After MT administration,the content of GSH increased significantly,the activity of GSH-Px increased significantly,and other indicators recovered to a certain extent,but there was no significant difference.It shows that MT has a certain alleviating effect on the oxidative stress of the five-parity gestation sows in restricted feeding.The number of white blood cells and lymphocytes were significantly reduced,the percentage of lymphocytes was significantly reduced,and the percentage of neutrophils was significantly increased.After MT administration,the number of white blood cells and neutrophils were significantly increased.The percentage decreased significantly,and the number of lymphocytes and the percentage of lymphocytes increased,but there was no significant difference.The content of CD3 and CD4 was significantly reduced,the content of CD8 was significantly increased,and CD4/CD8 was significantly reduced.After MT administration,each index recovered to a certain extent,indicating that MT can alleviate the decline in immune capacity of pregnant sows caused by restricted feeding.In summary,restricted feeding can cause CS in pregnant sows,serum CD3,CD4,CD8 content changes,the CD4/CD8 ratio decrease,and affect the immunity.Among them,the incidence of fifth-born pregnant sows is the highest,followed by first-born pregnant sows.In the rat CS model,MT can inhibit rat behavioral abnormalities,HPA axis disorders,histopathological changes,oxidative stress and immune dysfunction,and MT can correct the imbalance of mitochondrial dynamics in the spleen cells of CS rats and reduce internal stress.The unfolded protein response of the plasma reticulum and the weakening of the coupling of the mitochondrial-endoplasmic reticulum structure maintain the homeostasis of rat spleen cells,thereby protecting the spleen of CS rats.In the pig farm prevention test,MT can inhibit the CS of pregnant sows caused by restricted feeding,and play a certain role in alleviating changes in immune-related indicators of pregnant sows.It shows that MT is a potential drug to alleviate the CS caused by restricted feeding in pregnant sows and improve the immunity of pregnant sows. |
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