| Extreme weather and wet curtain cooling during the summer months can cause sudden cooling at high temperatures,which can lead to oxidative stress,tissue damage and reduced production performance.Perilla frutescens leaf is an inexpensive and readily available plant,and its extracts are effective in reducing oxidative stress in animals.However,the ability of Perilla frutescens leaf extract to alleviate oxidative stress and tissue damage caused by heat and cold stress in animals has not been reported.To address this issue,the following studies were carried out in this paper:Experiment 1: Construction of a sudden cooling stress at high temperatures model for mice.A total of 12 male KM mice of similar weight at 17 days of age were randomly divided into control and model groups,with 3 replicates of 2 mice each.The model group was given daily and regular sudden cooling stress at high temperatures(high temperature:35 ± 1℃,wind speed: 0.3 ～ 0.5 m/sfor 2 h;simulated wet-curtain stress: 15 ± 1℃,wind speed: 1.3 ～ 1.5 m/s for 3 h).Pre-feeding period 3 days,trial period 29 days.Food intake,water consumption,and body weight of mice were recorded during the test.Measurement of antioxidant parameters(T-SOD,GSH,MDA)and liver function parameters(ALT,AST,LDH)in mice at the end of the test.Morphological observations of the liver and various segments of the small intestine(duodenum,jejunum,and ileum)were also carried out.Determining the effects of sudden cooling stress at high temperatures on growth and development,biochemical parameters in serum,lung,and liver,and liver and intestinal morphology of mice.Experiment 2: In vitro antioxidant activity test of Perilla frutescens leaf extract.Gradient mass concentrations(0.1,0.3,0.6,0.9,and 1.2 mg/m L)of Perilla frutescens leaf extract,vitamin C,and two other plant extracts(orange peel extract and mugwort extract)were used to determine their scavenging capacity for DPPH,ABTS,and hydroxyl radicals.Based on the results,the in vitro antioxidant activity of Perilla frutescens leaf extract was evaluated,and its potential use for the mitigation of sudden cooling stress at high temperature was initially determined.Experiment 3: Alleviation effect of Perilla frutescens leaf extract by sudden cooling stress at high temperatures in mice.A total of 36 male KM mice of similar weight at the age of 17 days were randomly divided into 6 groups: control group,model group,and 4test groups according to the amount of Perilla frutescens leaf extract added(0.2,0.7,1.2and 1.7 mg/m L).Each group consists of 3 replicates,2 mice per replicate.Feeding management was the same as in experiment 1,with the model group and the 4 test groups being given regular daily sudden cooling stress at high temperatures,while the drinking water in the test groups was replaced with a solution of Perilla frutescens leaf extract at graded mass concentrations(0.2,0.7,1.2 and 1.7 mg/m L).Pre-feeding period 3 d,normal trial 29 d.Food intake,water consumption and body weight of mice were recorded during the test.At the end of the test,the antioxidant parameters(T-SOD,GSH,MDA)and liver function parameters(ALT,AST,LDH)were measured in the serum and liver of the mice,while morphological observations were made on the liver and various segments of the small intestine(duodenum,jejunum and ileum).The contents of each cecum segment were collected and analyzed for flora diversity using 16 S r RNA.The main test results are as follows:1.Successful establishment of a sudden cooling stress at high temperatures model in mice.The average daily food intake of the mice in the model group decreased and their body weight was significantly lower than that of the control group after the 15 th day of the test(P < 0.05).The MDA levels in the serum and liver of the mice in the model group were significantly higher than those in the control group(P < 0.05),and the T-SOD activity and GSH levels were significantly lower than those in the control group(P < 0.05).In addition,ALT,AST and LDH activity in the liver of mice in the model group were significantly increased(P < 0.05),and ALT and AST activity in the serum were significantly increased(P < 0.05).HE-stained sections of the liver showed that the number of pyknosis cells in the liver of the model mice was significantly higher than that of the control group(P < 0.05).The villi height and villi crypt ratio in the duodenum,jejunum and ileum of the model group were significantly lower than those of the control group(P <0.05).In summary,it can be concluded that relative to the normal control group,the model mice showed increased levels of oxidative stress in the serum and liver and damage to the liver and small intestine.2.Perilla frutescens leaf extract has in vitro antioxidant activity.Perilla frutescens leaf extract has various degrees of scavenging ability against DPPH,ABTS and hydroxyl radicals.The scavenging rate of DPPH radicals was significantly higher(P < 0.05)in Perilla frutescens leaf extract(0.3-1.2 mg/m L)than in orange peel and mugwort leaf extracts,with DPPH scavenging rate reaching 76.83% when the concentration of Perilla leaf extract was increased to 1.2 mg/m L.Perilla frutescens leaf extract can be used in subsequent studies of sudden cooling stress at high temperatures in mice.3.Perilla frutescens leaf extract mitigates the effects of sudden cooling stress at high temperatures in mice.(1)Effect of Perilla frutescens leaf extract on the alleviation of oxidative stress and hepatic impairment in sudden cooling stress at high temperatures model in mice: The mice in the test group fed with Perilla frutescens leaf extract recovered their feeding and body weight.Compared to the model group,the MDA content of 1.2mg/m L test group and 1.7 mg/m L test group decreased significantly(P < 0.05),T-SOD and GSH increased significantly(P < 0.05),and the viability of ALT,AST and LDH in the serum and liver had been significantly lower than that of the model group(P < 0.05);the number of pyknosis cells in the liver decreased significantly(P < 0.05).There was no significant difference in body weight,MDA,T-SOD and GSH in serum and liver,ALT,AST and LDH in serum and liver,and pyknosis cells number in liver between 1.2 mg/m L and 1.7 mg/m L test group mice compared to the control group(P > 0.05).In summary,Perilla frutescens leaf extract has some in vitro antioxidant activity and the addition of1.2-1.7 mg/m L of Perilla frutescens leaf extract can alleviate oxidative stress and liver damage in sudden cooling stress at high temperatures in mice.(2)Effect of Perilla frutescens leaf extract on intestinal morphology in mice subjected to sudden cooling stress at high temperatures: Compared to the model group,the intestinal villus height and villi crypt ratio of all small intestinal segments(duodenum,jejunum and ileum)were significantly higher in 1.2 mg/m L and 1.7 mg/m L test group(P < 0.05);compared to the control group,the intestinal villus height and villi crypt ratio of all small intestinal segments(duodenum,jejunum and ileum)were not significantly different in 1.2 mg/m L and 1.7 mg/m L test group(P > 0.05).(3)Effect of Perilla frutescens leaf extract on the intestinal microbiota in mice subjected to sudden cooling stress at high temperatures:Relative to the control group,a total of 102 intestinal genera were detected in the model group compared to the control group with a decrease of 16,and a significant distinction in flora structure,with a significant decrease in the relative abundance of the phylum Bacteroidota(P < 0.05)and a significant decrease in the relative abundance of the genera Bacteroides and Lactobacillus(P < 0.05).After feeding Perilla frutescens leaf extract,there was a trend towards recovery of the intestinal microbiota in the test groups.Compared to the model group,14,14,18 and 19 more genera could be measured in in the test groups at0.2,0.7,1.2 and 1.7 mg/ml,respectively.There was a clear distinction in the structure of the flora,with a significant increase in the relative abundance of the phylum Bacteroidota in test groups 0.7 and 1.2 mg/m L test group(P < 0.05),and a significant increase in the relative abundance of the genera Bacteroides and Lactobacillus in test groups 1.2 and 1.7mg/m L test group(P < 0.05).In conclusion,it can be concluded that the extract of Perilla frutescens leaf can alleviate the intestinal damage and intestinal microbiota disorder induced by sudden cooling stress at high temperatures in mice,and the combined effect of using 1.2-1.7 mg/m L of Perilla frutescens leaf extract is the best. |
