| ObjectiveThe effect and mechanism of spleen lymphocytes activated by Salviae miltiorrhizae+Radix puerariae were explored by microcalorimetry.The scientific connotation of synergistic effect of Salviae miltiorrhizae+Radix puerariae.MethodsTaking Danshen water extract,Pueraria water extract and Danshen + Pueraria water extract as research objects,the low,medium and high groups were set according to the dose concentration.Among them,the low,medium and high doses of water extract of Radix Salviae Miltiorrhizae and Radix Puerariae were: 250 μg/ml,500 μg/ml,1000 μg/ml;water extract of Salvia miltiorrhiza + water extract of Pueraria lobata was carried out according to 1:1.Compatibility,the low,medium and high dose groups are: 250 μg / ml,500 μg / ml,1000 μg / ml.They were applied to mouse spleen lymphocytes,and the growth and metabolism of mouse spleen lymphocytes were detected by microcalorimetry.The power-time curve was obtained and the thermodynamic parameters were fitted.The thermodynamic parameters: calorific value Q,thermal power P,peak time t,growth metabolic constant k for analysis;The effects of aqueous extract of Radix Salviae Miltiorrhizae,water extract of Radix Puerariae,water extract of Radix Salviae Miltiorrhizae and Radix Puerariae on the proliferation of mouse spleen lymphocytes were determined by CCK-8 method.Using Compu Syn 2.0 software to analyze the synergistic effect of Salvia miltiorrhiza water extract + Pueraria lobata water extract on spleen lymphocytes;Through the network pharmacology method,the Taiwan Traditional Chinese Medicine Database,the Chinese Medicine Active Ingredient Database,the therapeutic target database,the comprehensive database of human genes and gene phenotypes,and the mutual protein database for the active constituents and treatment of Salvia miltiorrhiza water extract and Radix Puerariae water extract The target and action pathways were analyzed by big data,and the related mechanisms of the aqueous extract of Radix Salviae Miltiorrhizae and the extract of Radix Puerariae on spleen lymphocytes were explored.ResultsThe microcalorimeter was used to monitor the effects of water extract of Salviae miltiorrhizae,Radix puerariae water extract,Salviae miltiorrhizae extract and Radix puerariae water extract on the growth and metabolism of spleen lymphocytes,and the thermogram curve was obtained to fit the corresponding thermodynamic parameters:（1）Heat production（Q）: The heat production（Q）of each control group of Salviae miltiorrhizae water extract,Radix puerariae water extract and Salviae miltiorrhizae water extract + Radix puerariae water extract group were 887.3030 J,820.7966 J and 853.6915 J,respectively;The caloric yield（Q）of the low,medium and high doses of Salviae miltiorrhizae was 1258.1397 J,1486.1937 J and 1531.5094 J,respectively;the caloric yield（Q）of the low,medium and high doses of Radix puerariae extract was 1028.8028 respectively.J,1178.0325 J,1112.6728J;the heat production（Q）of the low,medium and high dose groups of Salviae miltiorrhizae water extract + Radix puerariae water extract group were 1198.9540 J,1531.5094 J,2102.7189 J.（2）Peak heat production（P）: The peak heat production（P）of each control group of Salviae miltiorrhizae water extract,Radix puerariae water extract and Salviae miltiorrhizae water extract + Radix puerariae water extract group was 0.0982 m W,0.0835 m W,0.0819 m W.The peak heat production（P）of the low,medium and high doses of Salviae miltiorrhizae water extract was 0.1301 m W,0.1581 m W and 0.1879 m W,respectively.The peak heat production（P）of the low,medium and high doses of Radix puerariae extract was 0.1176 m W,0.1447 m W and 0.1826 m W,respectively.The peak heat production（P）of the low,medium and high dose groups of Salviae miltiorrhizae Water Extract + Radix puerariae water extract group were 0.1587 m W,0.1879 m W and 0.2294 m W,respectively.（3）Peak time（t）: water extract of Salviae miltiorrhizae,water extract of Radix puerariae,and water extract of Salviae miltiorrhizae+Radix puerariae,the peak time（t）of each control group of was 5651 s,5350s,4027 s.The peak time（t）of the low,medium and high doses of Salviae miltiorrhizae extract was 5784 s,5736s and 5491 s,respectively.The peak time（t）of the low,medium and high dose groups of Radix puerariae water extract was 5359 s,5628s and 5066 s,respectively.The peak time（t）of the low,medium and high doses of Water Extract of Salviae miltiorrhizae+Radix puerariae was 3886 s,3955s and 4221 s,respectively.（4）Growth rate constant（k）: growth rate constant（k）of each control group of Salviae miltiorrhizae water extract,Radix puerariae water extract and Salviae miltiorrhizae+Radix puerariae water extract group was 0.01350s-1,0.01365s-1,0.01358s-1.The growth rate constants（k）of the low,medium and high doses of Salviae miltiorrhizae aqueous extract was 0.0181 s-1,0.0305 s-1,0.0325 s-1.The growth rate constants（k）of the low,medium and high doses of Radix puerariae water extract was 0.0190s-1,0.0206s-1,0.0280s-1,respectively.The growth rate constants（k）of the low,medium and high dose groups of the water extract of Salviae miltiorrhizae and the water extract of Radix puerariae was 0.0183s-1,0.0353s-1 and 0.0375s-1,respectively.（5）Water extract of Salviae miltiorrhizae,water extract of Radix puerariae,water extract of Salviae miltiorrhizae and water extract of Radix puerariae all increased the heat production of spleen lymphocytes growth and metabolism,increased the maximum heat power,decreased the peak time,increased the growth rate constant,and have a certain dose-effect relationship.The effects of water extract of Salviae miltiorrhizae and Radix puerariae on water extract were higher than those of Salviae miltiorrhizae and Radix puerariae.The high-dose group combined with the heat production,the maximum heat power was the highest,the peak time was the shortest,and the growth rate constant was the largest.（6）CCK-8 method was used to detect the proliferation of spleen lymphocytes in mice by water extract of Salviae miltiorrhizae,water extract of Radix puerariae,water extract of Salviae miltiorrhizae and water extract of Radix puerariae.The results showed that the spleen lymphocytes reached the optimal state in 2 hours,which was consistent with the peak heat generation time of the cells detected by microcalorimetry.At the same time,the administration groups could promote the proliferation of spleen lymphocytes compared with the control group.The water extract of Salviae miltiorrhizae and the aqueous extract of Radix puerariae had the greatest proliferative effect relative to each group.（7）Using Compu Syn 2.0 software to analyze the effect relationship of Salviae miltiorrhizae water extract Radix puerariae extract group,the combined low,medium and high index of combined administration group were 0.044,0.376,0.418,respectively.All shows good synergy.（8）Using the network pharmacology method,the data analysis of the possible mechanism of the aqueous extract of Salviae miltiorrhizae and the extract of Radix puerariae showed that p53 signaling pathway,TNF signaling pathway,PI3K-Akt signaling pathway and T cell receptor signal Pathways may be the basis for their synergy.ConclusionsBoth Radix puerariae and Salviae miltiorrhizae can induce the activation of mouse spleen lymphocytes,and the combined effect of the two can synergistically promote the activation of mice spleen lymphocytes,and the combined administration effect is better than the single administration group.The 20 pathways in which Radix puerariae+Salviae miltiorrhizae may play a role in promoting cell proliferation and positively regulating the metabolism of spleen lymphocytes,thereby activating spleen lymphocytes and ultimately playing a synergistic role. |
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