Effects Of Tea Tree Oil On Lipid Deposition In Bovine Hepatocytes Induced By Palmitic Acid And Health Status Of Bovine During Late Perinatal Period

The incidence of fatty liver in bovine during perinatal period is high,leading to a significant decrease in milk yield and even other diseases.At present,the treatment cost of this disease is high and the effect is unsatisfactory,so it is particularly important to prevent the occurrence of fatty liver in bovine.Tea tree oil（TTO）,as one of the natural essential oils,has antibacterial,anti-inflammatory,antioxidant and immune-enhancing effects.There are still few studies on lipid metabolism and ruminant,and the specific mechanism and function are still to be studied and explored.In this study,palmitic acid（PA）was used to induce lipid deposition in dairy hepatocytes to investigate the effects of TTO on PA induced lipid deposition in dairy hepatocytes,and to investigate the effects of TTO in dairy diet on performance and serum biochemical indices in late perinatal period.1.Effects of palmitic acid on lipid deposition in bovine hepatocytesTo investigate the concentration and treatment time of PA on hepatocyte lipid deposition,the bovine hepatocytes were treated with 50,100 and 200 μM PA for 2 d,3 d and 4 d,and the intracellular contents of each indicator were detected using ATP,TG and VLDL kits.The results showed that:treated with 100 and 200 μM PA for 3 days,the intracellular ATP content was significantly increased（P<0.05）;Treatment with 100 μM PA for 3 d and 4d,the intracellular TG content was significantly increased（P<0.05）;The intracellular VLDL content of hepatocytes was significantly decreased after treatment with 100 μM PA for 3 d and 4 d（P<0.05）.Based on these results,bovine hepatocytes were stimulated withl 00 μM PA for 3 days in subsequent experiments.2.Effect of tea tree oil on lipid metabolism of bovine hepatocytes in palmitic acid stimulated bovine hepatocytesTo investigate the effect of TTO on lipid metabolism of dairy bovines in PA stimulated bovine hepatocytes,in this experiment CCK8 and TG kits were used to detect the effects of TTO treatment with different concentrations（0.001562,0.003125,0.00625,0.0125,0.025,0.05 and 0.1%）on cell proliferation toxicity and changes in intracellular TG content of primary hepatocytes.The results showed that:0.00625%did not impair cell viability of bovine hepatocytes and elicited a remarkable decrease in TG content.Therefore,0.00625%TTO is a potential treatment option for reducing lipid accumulation induced by PA in bovine hepatocytes.The gene expression levels of SREBP1c,FAS,ACC1,PPAR-α,CPT1A and CPT2 were detected by qRT-PCR.The protein expression levels of SREBP1c and FAS were detected by Western Blot.The results showed that TTO could significantly down-regulate the gene expression of SREBPlc,FAS and ACC1 and the protein expression of SREBP1c and FAS（P<0.05）.The expression of genes related to fatty acid oxidation-related genes,such as PPAR-α,CPT1A and CPT2,were significantly up-regulated（P<0.05）.3.Effects of tea tree oil on inflammation,oxidative stress and endoplasmic reticulum stress in bovine hepatocytes stimulated with palmitic acidTo explore the effects of TTO on inflammation,oxidative stress and endoplasmic reticulum stress in bovine hepatocytes stimulated with PA,the expression genes related to inflammation and endoplasmic reticulum stress were detected by qRT-PCR.Protein expression levels of p-p65 and p-IκB were detected by Western Blot.Intracellular ROS levels and mitochondrial membrane potential were detected by flow cytometry using reactive oxygen species detection kit and mitochondrial membrane potential kit.The results showed that TTO significantly decreased the gene expression of IL-6 and TNF-α（P<0.05）,and NF-κB-related proteins p-p65 and p-IκB（P<0.05）.TTO significantly decreased intracellular ROS levels and mitochondrial membrane potential（P<0.05）,and significantly decreased the expression levels of endoplasmic reticulum stress-related genes GPR78,ATF4,ATF6,sXBP1,CHOP,EIF2A,ASK1 and HSP70（P<0.05）.4.Effects of tea tree oil on performance and serum biochemical indices of dairy bovine in late perinatal periodIn order to explore the effect of TTO on the production performance and serum biochemical indexes of perinatal cows,24 healthy late perinatal Holstein bovines with similar birth dates were randomly divided into 3 groups of 8 bovines.The control group was fed with the basal diet,and two experimental groups were supplemented with 1 g/kg and 2 g/kg TTO（dry matter basis）on the basis of the control group,namely LTTO group and HTTO group,respectively.The experiment lasted for 21 days.The results showed as follows:Compared with the control group,there were some differences in the results of the LTTO group and the HTTO group:Dry matter intake（DMI）in the LTTO group showed an upward trend（P=0.07）,the LTTO group and HTTO group showed an upward trend in 4%standard milk content（P=0.09）,and the Albumin（ALB）content in the LTTO group showed an upward trend（P=0.08）,the LTTO group significantly increased the content of immunoglobulin G（IgG）in the serum（P<0.05）;it is worth noting that the HTTO group significantly decreased the activity of aspartate aminotransferase（AST）（P<0.05）,the LTTO group and the HTTO group significantly increased the content of glucose（GLU）in the serum（P<0.05）,and the triglyceride（TG）content showed a downward trend（P=0.09）.In summary,1-2 g/kg TTO can maintain liver function and improve immunity of dairy bovines.The above results suggest that（1）TTO can not only alleviate the lipid deposition induced by PA,but also alleviate the oxidative stress and endoplasmic reticulum stress of inflammatory reaction.（2）The supplementation of 1-2 g/kg TTO in diets of dairy bovines during late perinatal period can improve immunity and maintain liver function...