Investigating The Biological Essence Of Spleen Deficiency Syndrome-Buzhong Yiqi Decoction From The Molecular Mechanism Of Aging

Based on the connotation of the relationship between prescriptions and syndromes,the"Relevance of prescription and syndrome"(RPS)has been one of the most important academic proposition in the study of Formulalogy.Network Pharmacology(NP)is a new method to explore the inner biological relationship between traditional Chinese medicine(TCM)formula and syndrome.Spleen deficiency syndrome(SDS)is closely related to aging in TCM.However,there are few research that focused on exploring the biological molecular mechanism of SDS-aging.According that,based on RPS,we investigated the pharmacological molecular network of Buzhong Yiqi Decoction(BYD)by NP with the logical basis of the high SDS-B YD relevance and the argument of aging.Based on above,we dynamically observed the biological molecular characteristics of aging-like changes during SDS rat model development,as well as the pharmacological mechanism of BYD.This paper is divided into two parts:literature review and experimental study.The literature review mainly involves three aspects as follows:the research progress of RPS,the research progress of SDS-aging relevance,and the modern research progress of BYD.The experimental study mainly includes three parts:"Investigating the biological relevance between SDS and BYD by NP","Investigating the evolutionary relevance between SDS and aging",and"Investigating the biological mechanism of SDS and BYD from aging".Study 1:Investigating the biological relevance between Spleen deficiency syndrome and Buzhong Yiqi Decoction by Network Pharmacology.Methods:The active ingredients and related targets of BYD(Astragalus,Atractylodes macrocephala,Cimicifuga,tangerine peel,bupleurum,ginseng,liquorice,angelica,ginger,jujube)were searched respectively on TCMSP platform.The PPI network was established on STRING platform and then imported into Cytoscape.After clustering PPI network with MCODE plugin to extract core target clusters,the ClueGO plugin was used to target core clusters.Finally,Go and KEGG enrichment analysis were carried out to extract the main physiological function and bio-molecular signaling pathway.Results:I.143 kinds of active components of BYD were screened and 162 related targets were found,among which 155 targets had PPI relationship and 9 core clusters were obtained by clustering.II.13 GO groups were enriched,including nitric oxide biosynthesis,promoting smooth muscle cell proliferation,etc.12 KEGG groups were enriched,which involved AGE-RAGE signaling pathway,IL-17 signaling pathway,relaxin signaling pathway,etc.Conclusion:The correlated mechanism of SDS-BYD might be related to the inflammatory regulation process,which was led by AGE-RAGE signaling pathway,IL-17 signaling pathway and relaxin signaling pathway by mediating NF-κB.Study 2:Investigating the evolutionary relevance between Spleen deficiency syndrome and aging.Methods:Wistar rats were randomly divided into control group and model group,each of which contained 18 rats.The control group was fed regularly,and the model group was reconstructed the SDS model by "exhausted swimming+food control".Rats in each group were:I.observed the appearance behavior score every other day,weighed the food intake every day,and observed the body mass and fecal moisture content at the end of each week.II.At the end of the 2nd-4th weeks,the forced load swimming experiment was performed to measure the duration of load swimming;at the 3rd and 4th week,the Morris Water Maze experiment was carried out to measure the swimming speed,spatial probe-lantcncy,platform crossings,distance ratio of target quadrant,and explored route.ⅢOn the 14th,21st and 28th day of the experiment,D-xylose solution was feed by gavage,then the urine was collect to measure the D-xylose content by ELISA.On the 15th,22nd and 29th day of the experiment,three batches of anesthesia were randomly divided to kill:Spleen and thymus were weighed to calculate organ index.Serum was taken to measure the GAS,MTL,AGEs,BDNF,VIP,IL-1β,IL-6,TNF-α levels by ELISA,and measure the CRP level by biochemical process.The hippocampus was taken to measure the levels of AGES,Aβ1-40,Aβ1-42,glutamate,GABA,IL-1β,IL-6 and TNF-α by ELISA.Results:Compared with the control group,the rats in the model group showed changes as follows:I.The scores of skin-hair,behavior,activity and sleep state increased significantly(P<0.01)in the second to fourth weeks of the experiment;The body weight and food intake decreased significantly(P<0.01)in the first to fourth weeks of the experiment;The fecal water content increased(P<0.05)in the third and fourth weeks.II.In the fourth week of the experiment,the weighted swimming time decreased significantly(P<0.01);In the third and fourth weeks,the latency of space exploration increased,the number of crossing platform decreased,and the distance ratio of target quadrant decreased(P<0.05).Ⅲ.The excretion rate of D-xylose in urine decreased significantly(P<0.01)and the level of serous VIP decreased in the second to fourth weeks of the experiment;The level of serous GAS decreased in the third and fourth weeks;the level of serous MTL decreased in the fourth week of the experiment(P<0.05 or 0.01).Ⅳ.The levels of GABA,IL-1β,Aβ1-40,BDNF,ages,Aβ1-42/Aβ1-40,glu,IL-6 and TNF-α in hippocampus were significantly increased(P<0.05 or 0.01).V.The serous levels of IL-6 and TNF-α were both increased in the second week(P<0.05 or 0.01),and decreased significantly in the fourth week(P<0.01).Conclusion:In SDS model,Brain aging,appeared as memory deficit,was gradually formed during SDS model copying,the molecular features of which involved toxic protein accumulation,inflammation level increase,and exciting imbalance of glutamate-GABA in hippocampus.Study 3:Investigating the biological mechanism of Spleen deficiency syndrome and Buzhong Yiqi Decoction from aging.Methods:Wistar rats were randomly divided into control group,model group,low dose TCM group(L group)and high dose TCM group(H group),each of which contained 8 rats The control group was fed regularly,the model group and the two TCM groups were made into models for 4 weeks according to the method of study 2.Besides that,the low and high dose administration groups were given 5.85g/kg/d and 17.55g/kg/day respectively from the 15th to 28th day.For rats in each group:I.The appearance behavior score,food intake,body mass and fecal moisture content were observed as the time and method of Study 2.Ⅱ.According to the method of the second study,the forced load swimming experiment was performed at the end of the fourth week,and the Morris Water Maze experiment was performed at the fourth week.III.D-xylose solution was infused on the 28th day of the experiment,and the urinary excretion rate of D-xylose was determined as the method of Study 2.On the 29th day,the rats were killed in anesthesia:the spleen and thymus were weighed to calculate the organ index;the serum was taken to measure the levels of GAS,MTL,BDNF,VIP,IL-1 β,IL-6,TNF-α;the hippocampus was taken to measure the levels of AGEs,Aβ 1-40,Aβ1-42,Glu,GABA,IL-1β,IL-6,TNF-α TGF-β1;the immuno-histochemistry was used to measure the levels of p65 NF-κB,p-p65 NF-κB,and Western blot were used to measure the levels of AGEs,Aβ1-40,Aβ1-42,Glu,GABA,IL-1β,IL-6,TNF-α and TGF-β1.The expression of NF-κB.RAGE and Iba-1 was detected by immuno-fluorescence double standard method.The co-expression of NeuN+cleared Caspase-3,Iba-1+TNF-α,and Iba-1+TGF-β1 was detected by immuno-fluorescence double standard method.The expression of p65 NF-κB,p-p65 NF-κB and Iba-1 was detected by immuno-histochemistry and Western blot.Results:① Compared with the control group,rats in the model group:a.Skin-hair,behavior state,activity degree,sleep state scores were all increased(P<0.05 or 0.01),body mass and food intake were both decreased significantly(P<0.01),fecal water content was increased(P<0.05).b.The time of weight-bearing swimming was decreased significantly(P<0.01);The excretion rate of urinary D-xylose,the level of serous GAS and MTL were decreased significantly(P<0.01);The level of serous VIP was increased significantly(P<0.01).c.The spatial lantency was increased significantly(P<0.01),the distance ratio of target quadrant was decreased(P<0.05),and the exploration route was changed to a random strategy.d.Serous BDNF level was decreased(P<0.05);Hippocampal AGEs,Aβ1-42,Aβ1-42/Aβ1-40,RAGE,glu,Iba-1,IL-1β,IL-6,TNF-α level were all increased(P<0.05 or 0.01);The expression and activation of hippocampal p65 NF-κB were both increased(P<0.05 or 0.01);The GABA,TGF-β1 level in hippocampus were decreased(P<0.05 or 0.01);The co-expression of NeuN and cleared caspase 3,Iba-1 and TNF-α were both increased(P<0.05 or 0.01).e.The expression of Iba-1,p65 NF-κB and its activation were significantly increased(P<0.01),and the expression of GnRH was significantly decreased(P<0.01)in hypothalamus.f.The index of spleen and thymus decreased significantly(P<0.01),and the levels of serous IL-1β,TNF-α and TGF-β1 were decreased significantly(P<0.01).Ⅱ.Compared with the model group,the rats in each TCM group were changed as follows:a.The general behavior score,body mass,food intake and fecal water content were all improved in varying degrees(P<0.05 or 0.01).b.The weighted swimming time was increased significantly(P<0.01);The urinary excretion rate of D-xylose,the level of serous GAS and MTL were increased(P<0.05 or 0.01);the level of serous VIP was decreased significantly(P<0.01).c.The spatial lantency was significantly shortened(P<0.01),and the route of space exploration was changed to a trended strategy;Besides that,the platform crossings was increased in H group(P<0.05).d.The level of serous BDNF was increased(P<0.05);The co-expression of NeuN+cleaved caspase 3,Iba-1+TNF-α were both decreased(P<0.05);the co-expression of Iba-1+TGF-β1 was increased(P<0.05 or 0.01)in hippocampus;the expression and activation of hippocampal p65 NF-κB was decreased(P<0.05 or 0.01);the levels of hippocampal AGEs,Aβ1-42,RAGE,glu,Iba-1 and IL-1 β level were all decreased(P<0.05 or 0.01).Besides that,the level of IL-6 and TNF-α in hippocampus in L group were significantly decreased(P<0.01),and the levels of hippocampal TGF-β1 were significantly increased(P<0.01).e.The expression of Iba-1,p65 NF-κB and their activation in hypothalamus were all decreased(P<0.05 or 0.01),and the expression of hypothalamic GnRH was increased(P<0.05).f.The thymus index,serous IL-1 β and TNF-α levels in H group were all increased(P<0.05 or 0.01).The serous TGF-β1 level in two TCM groups were both increased(P<0.05 or 0.01)and the serous IL-6 level were significantly decreased(P<0.01)in each group.Conclusion:There existed both hippocampal inflammatory aging and peripheral immune aging in SDS rats.It might be the M1 microglia polarization and neuron apoptosis those caused by the activation of AGEs/RAGE/NF-κB signaling pathway that was the potential mechanism of brain aging in SDS rats.BYD showed good effect against both SDS and aging,the mechanism of which might involve the neuroprotection effect with the inhibition of AGEs/RAGE/NF-κB signaling pathway and the M2 polarization of microglia.In this study,based on the mechanism of aging,we combined the RPS theory and NP method to investigate the BYD-SDS biological mechanism in system-organ-molecule level,which could provide a new direction for exploring the biological basis of RPS.