Study On The Anti-inflammatory Effect, Pharmacodynamic Material Basis And Mechanism Of Action Of Simplified Zhiqin Decoctio

BackgroundThe Simplified Zhi Qin Decoction is derived from Zhzi Huangqin Decoction,a surgical monograph written by Li Xun in the Southern Song Dynasty.Zhzi Huangqin Decoction is composed of 12 herbs with the effects of clearing heat and detoxifying,cooling blood,and benefiting qi,etc.It is used to treat"fever after the ulceration of back sores due to dietary injury and improper regulation.The Simplified Zhi Qin Decoction whole formula is simplified to 4 herbs of Forsythiae Fructus,Vaccariae Semen,Gardeniae Fructus and Gardeniae Fructus,with effects of draining wind and clearing heat,dipping fire and detoxicating,subduing swelling and promoting lactation,and diuretic and laxative,mainly for the treatment of pediatric Pneumonia,female mastitis,etc.The Simplified Zhi Qin Decoction has been clinically applied for many years as a hospital agreement prescription with remarkable efficacy,but there is a lack of systematic pharmacodynamic studies and the mechanism of action is still unclear.The purpose of this paper is to conduct the screening of the optimal ratio of the anti-inflammatory effect of the Simplified Zhi Qin Decoction,the validation of the anti-inflammatory effect in animal tests and the elucidation of its mechanism of action,so as to provide a test basis for the development of the hospital formulation of this formula.This study was funded by Chongqing Key Construction Discipline of TCM Rehabilitation（2021-4322190044）and Chongqing Science and Health Joint TCM Science and Technology Project（No.ZY201802041）.Purposes1.To evaluate the anti-inflammatory efficacy of the Simplified Zhi Qin Decoction.2.To explain the anti-inflammatory effects mechanism of the Simplified Zhi Qin Decoction.Methods1.The method for studying the anti-inflammatory effects of different ratios of the Simplified Zhi Qin Decoction.（1）Group administration and inflammation model replication in rats:100μg_·m L^-1 lipopolysaccharide solution was injected intraperitoneally to replicate the inflammation model.Rats were randomly divided into17 groups:control group,model group and the Simplified Zhi Qin Decoction group with different ratios.The Simplified Zhi Qin Decoction solution or saline was administered for 7 d.After the last administration,lipopolysaccharide was injected intraperitoneally into the model except for the control group which was injected with equal amount of saline intraperitoneally.（2）Observation indexes and detection methods:Observe the physical appearance,voluntary activities,fecal status and color of rats in each group,and detect the body temperature.Blood was collected to measure routine blood indicators.The serum TNF-α,PGE₂,IL-1βand IL-6 levels were detected.The general body condition,body temperature,blood routine and inflammatory factors were used as indicators,and orthogonal analysis was used to obtain the best ratios for subsequent experiments.2.The Simplified Zhi Qin Decoction to treat inflammation of the pharmacodynamics and its mechanism of action research method.（1）Group administration and inflammatory model mice replication:100μg_·m L^-1lipopolysaccharide solution was injected intraperitoneally to replicate the inflammation model.Mice were randomly divided into 6 groups:control group,model group,dexamethasone group and the Simplified Zhi Qin Decoction 1 g_·kg^-1,2 g_·kg^-1,4 g_·kg^-1group.After 7 d of administration,lipopolysaccharide was injected intraperitoneally into the model except for the control group,which was injected with equal amount of saline intraperitoneally.（2）Efficacy observation indexes and test methods:observe the appearance of mice,body temperature,water consumption,voluntary activities and urine volume were detected.Blood was taken to measure blood routine.The lung,liver,thymus and spleen were taken,and the lung,liver,thymus and spleen organ indices and their wet/dry weight ratios were calculated,and the pathological morphology of the organs were observed by HE staining.The NO,PGE₂,TNF-α,IL-1β,IL-6 in serum and urine and serum Ig G,Ig M and Ig A contents were measured.（3）Mechanism of action observation indexes and detection methods:Immunohistochemistry was used to detect TNF-αexpression in lung tissue,and Western bolt was used to detect the expression of key proteins of JAK2/STAT,PI3K/AKT and TNF pathway in lung tissue.3.Network pharmacological prediction the Simplified Zhi Qin Decoction for the treatment of inflammation main pharmacological components and targets and molecular mechanisms.The TCMSP database was used to screen the four active ingredients of the Simplified Zhi Qin Decoction and their corresponding targets,and other databases were used to supplement the unpredicted preferred ingredients and their targets.Using"inflammation"as the keyword,we searched the Gene Cards database for potential targets of inflammation,supplemented with DRUGBANK,OMIM,CTD and TTD databases,and combined and de-duplicated to obtain inflammation targets.The active ingredient targets and inflammatory targets were taken to intersect to get the Simplified Zhi Qin Decoction anti-inflammatory targets.the STRING database was constructed for protein network interactions,and the core targets were screened.Key targets GO functional annotation and KEGG pathway enrichment analysis.Molecular docking was conducted to investigate the binding degree of main active ingredients and key targets.Results1.The optimal ratio of the Simplified Zhi Qin Decoction anti-inflammatory effect is 4:1:4:2.Compared with the control group,the rats in the model group had rough hair,poor mental status,reduced voluntary activities and diarrhea;body temperature was significantly increased;blood routine WBC,Mon%,Gran,Gran%were significantly increased,Lymph,Lymph%,PLT,PCT were significantly decreased;serum TNF-α,PGE₂,IL-1β,IL-6 levels were significantly increased（all P<0.05）.Compared with the model group,rats in 5-9 and 13-16 groups of the Simplified Zhi Qin Decoction had flat and smooth hair,recovered mental status,increased voluntary activities and improved diarrhea symptoms,and the optimal ratio was 8:3:6:4;the body temperature of rats in 2-5,6,9,11 and 13-15 groups were significantly reduced at 2.0-7.0 h,and the optimal ratio was 4:0:2:3;WBC in 2-16 groups were significantly decreased,Mon%in 5,7-9,11 and 14 groups were significantly decreased,Gran in 3,4,7-11,13 and 15 groups were significantly decreased,Gran%in 2 and 15 groups were significantly decreased,Lymph in 5,6 and 15 groups were significantly increased,Lymph%in 2,11 and 15 groups were significantly increased,and the optimal ratio was4:1:4:2;Serum TNF-α,PGE2,IL-1βand IL-6 in rats in 2-16 groups were significantly reduced,and the optimal ratio was 4:1:4:2（all P<0.05）.The best ratio of the anti-inflammatory effect of the Simplified Zhi Qin Decoction was Forsythiae Fructus:Vaccariae Semen:Gardeniae Fructus:Scutellariae Radix=4:1:4:2.2.The Simplified Zhi Qin Decoction had significant anti-inflammatory effect on lipopolysaccharide-induced inflammatory mice.Compared with the control group,Mice in the model group had rough hair,poor mental status,significantly increased body temperature,significantly reduced voluntary activities,significantly increased water consumption and significantly reduced urine output;the wet/dry weight ratios of lung and liver significantly increased,the lungs,liver,thymus and spleen showed pathological damage;WBC,Mon%,Gran,Gran%of blood routine were significantly increased,RBC,HGB,Lymph,Lymph%,PLT,PCT were significantly reduced;serum IL-6 was significantly increased,serum and urine No,TNF-α,IL-1β,PGE₂ were significantly increased,and serum Ig G,Ig A,Ig M were significantly decreased（all P<0.05）.Compared with the model group,the Simplified Zhi Qin Decoction 1 g_·kg^-1 group,2 g_·kg^-1 group and 4 g_·kg^-1 group of inflammatory mice with the best ratio of the Simplified Zhi Qin Decoction had flat and smooth hair,recovered mental status,and significantly decreased body temperature.The wet/dry weight ratios of lung and liver in the Simplified Zhi Qin Decoction 1 g_·kg^-1 group,2 g_·kg^-1 group and4 g_·kg^-1 group were significantly decreased（all P<0.05）,and pathological damage of the lung,liver,thymus,and spleen was reduced.The Simplified Zhi Qin Decoction 1g_·kg^-1 group had significantly lower WBC,significantly higher RBC,Lymph%,HGB,PLT;the Simplified Zhi Qin Decoction 2 g_·kg^-1 group had significantly lower WBC,Mon%,Gran%,Lymph significantly increased,serum IL-6 and urinary TNF-α,PGE2significantly decreased;the Simplified Zhi Qin Decoction 4 g_·kg^-1 group RBC,PLT,PCT significantly increased,serum IL-6 levels significantly decreased,the No,TNF-α,PGE₂,IL-1βin serum and urine were significantly decreased,and serum Ig G,Ig M,Ig A levels were significantly increased（all P<0.05）.3.Network pharmacological analysis obtained 27 main active ingredients of the Simplified Zhi Qin Decoction anti-inflammatory effect and 30 key targets of anti-inflammatory effect,including JAK2/STAT,PI3K/AKT,NF-κB,TNF pathway proteins.Sixty-six major active ingredients were obtained,including 27major active ingredients such as phillyrin,vaccarin,geniposide,baicalin,quercetin,β-sitosterol,kaempferol,etc.;30 key targets were involved in the anti-inflammatory effect of the Simplified Zhi Qin Decoction,including JAK2/STAT,PI3K/AKT,NF-κB,TNF pathway proteins.Molecular docking indicated that the main active ingredient docked with key targets with a score>4.25 accounting for 52.09%of the total.4.Animal tests demonstrated that the anti-inflammatory mechanism of the Simplified Zhi Qin Decoction was able to significantly reduce the expression of JAK2/STAT-PI3K/AKT-NF-κB-TNF signaling pathway proteins.Compared with the control group,the expression of TNF-αin lung tissues of mice in the model group was significantly increased;the expression levels of JAK2,p-JAK2,p-STAT5,STAT3,p-STAT3,PI3K,AKT,p-AKT,NF-κB p65,TNF-α,PTGS2 proteins in lung tissues were significantly increased（all P<0.05）.Compared with the model group,TNF-αwas significantly decreased in the lung tissue of the Simplified Zhi Qin Decoction 4 g_·kg^-1group;the expression levels of p-JAK2,p-STAT5,STAT3,AKT,NF-κB p65,PTGS2were significantly reduced in the Simplified Zhi Qin Decoction 1 g·kg^-1 group,the expression levels of p-STAT5,AKT,TNF-α,PTGS2 were significantly reduced in the Simplified Zhi Qin Decoction 2 g_·kg^-1 group,the expression levels of the JAK2,p-JAK2,p-STAT5,p-STAT3,STAT3,PI3K,p-AKT,AKT,NF-κB p65,and PTGS2 of Simplified Zhi Qin Decoction 4 g_·kg^-1 group were significantly decreased（all P<0.05）.Conclusion1.The optimal ratio of the anti-inflammatory effect of the Simplified Zhi Qin Decoction on lipopolysaccharide-induced inflammatory rats is 4:1:4:2 for Forsythiae Fructus:Gardeniae Fructus:Scutellariae Radix.2.The Simplified Zhi Qin Decoction showed significant anti-inflammatory effects on lipopolysaccharide-induced inflammatory mice.The main pharmacodynamic components of the anti-inflammatory effects were 27,including phillyrin,vaccarin,geniposide,baicalin,quercetin,β-sitosterol,kaempferol,etc.3.Network pharmacology prediction results suggest that the anti-inflammatory effect of the Simplified Zhi Qin Decoction involves 30 key targets,including JAK2/STAT,PI3K/AKT,NF-κB and TNF pathway proteins.4.Animal experiment validation showed that the anti-inflammatory effect of the Simplified Zhi Qin Decoction was associated with reduced the expression levels of JAK2/STAT-PI3K/AKT-NF-κB-TNF pathway proteins.