Xing Wang,Tian-Tian Deng,Xia Li

1Beijing University of Chinese Medicine,Beijing 100029,China;2Dongzhimen Hospital Beijing University of Chinese Medicine,Beijing 100700,China.

Background:To explore the potential mechanism of the classic ancient prescription Qinghaobiejiatang (QHBJT)in treating renal interstitial fibrosis.Methods: Obtain the active ingredients of Qinghao (Artemisiae annuae herba),Biejia (Trionycis carapax),Dihuang (Rehmanniae radix),Zhimu (Anemarrhenae rhizoma),and Mudanpi (Moutan cortex) through TCMSP and TCMID databases.Collect disease targets through the GENECARDS database.Use Venny2.1.0 platform to draw Venn diagrams to map drugs and disease targets.Import the key targets into Cytoscape 3.7.2 software to draw a network diagram of “drugs-active ingredients-diseases-key targets”.The STRING11.0 database was used to construct the key target protein interaction network diagram.Use R language for Gene ontology function and kyoto encyclopedia of genes and genomes pathway enrichment analysis.Results:A total of 317 active ingredients were obtained through screening,involving 166 targets,and 102 active ingredients related to disease targets,mainly involved in the regulation of key targets such as FOS,IL6,MTOR,MAPK8,RELA,CCND1,TP53,EGFR,and CASP3 signal pathways related to viral infection,tumor-related,apoptosis,signal transduction,fluid shear stress and atherosclerosis play a synergistic role in the treatment of renal fibrosis.Conclusion:The effect mechanism of QHBJT in treating renal interstitial fibrosis is related to inflammation,oxidative stress,hypoxia,apoptosis,pathological activation and damage of renal tubular epithelial cells mediated by the above pathways.

Key words: Internet pharmacology,Qing-Hao-Bie-Jia-Tang,Renal interstitial fibrosis,Mechanism research,Latent pathogen

Background

Chronic kidney disease (CKD) is a chronic disease characterized by a gradual and irreversible loss of kidney function.A study in 2012 found that the incidence of CKD in the Chinese population was 10.8%,and WHO announced the case fatality rate of CKD was 1.5% 1.So CKD has gradually become a global health problem.Renal interstitial fibrosis (RIF)plays an important role in the pathogenesis and progression of CKD.At present,the formation mechanism of RIF is not fully understood.It is generally believed that it is related to the disorder of extracellular matrix (ECM) synthesis,which leads to the destruction of renal tissue structure and renal failure.

“Fuxie” is a characteristic theory of traditional Chinese medicine.It was first seen in theHuangdi Nei Jing(written by the legendary Yellow Emperor in the Western Han Dynasty): “Sickness in Winter is Caused by Cold,and Cold in Spring.” It means that you feel cold in the previous season and get sick in the next season.Zhang Zhongjing’sTreatise on Febrile Diseases(written by Zhang Zhongjing in the end of the East Han Dynasty) first appeared in the concept of “Fuqi”.In the Ming and Qing dynasties,a complete academic system was formed and became an important part of “Fuxie” and “Wen Disease”.It is often used clinically to guide the treatment of difficult diseases,such as chronic bacterial infections 2,chronic viral infections 3,autoimmune diseases 4,tumors 5,etc.Professor Li Xia believes that renal interstitial fibrosis can be treated with “Fuxie” theory.Modern research believes that renal interstitial fibrosis is related to a large number of noxious stimuli,such as trauma,infection,metabolic disorders,inflammation,etc.,which repeatedly and permanently drive fibrosis,leading to excessive deposition of extracellular matrix and destroying normal tissue structure 6.This is consistent with the pathogenic characteristics of “Fuxie’s” “feeling but not sending out” and or “recurring”,so it belongs to the category of “Fuxie” 7.Professor Wu Xiongzhi believes that the main reason why evil is lurking is that there is insufficient righteousness and no contention between righteousness and evil.Suwen·Yin-Yang Yingxiang Theory(written by the legendary Yellow Emperor in the Western Han Dynasty) says,“If you don’t store essence in winter,you must be ill in spring”.Due to the deficiency of the righteous and the abundence of evil,the “Evil Qi” stays in the “Three Yin” ;due to the deficiency of the spleen and kidney and the dysfunction of the three energizers,the kidney’s function of distinguishing and secreting turbidity is reduced.The damp turbidity and the poison must not be leaked.It willaccumulate in the body and blood.Phlegm and blood stasis combine to form a tangible disease nest,and toxins are added to endogenously,which together block the Jueyin collaterals and become fibrotic over time.After renal interstitial fibrosis,the blood vessels are reduced,making it difficult for drugs to reach the focus.The “Evil Qi” is lurking in the renal interstitium,and the evil that stays in the “Yin” part is accumulating.The “Evil Qi” gradually increases,leading to fatigue and subsequent kidney failure.Suwen·Yin and Yang Yingxiang Dalunsays,“If you get typhoid fever in winter and it is not cured,febrile disease occurs in spring”. “Fuxie” easily transforms into “Fire-pathogen”.Suwen·Liu Wei Zhi Da Lun(written by the legendary Yellow Emperor in the Western Han Dynasty) says that “Shaoyangzhishang,Huoqizhizhi”.It is said that the transformation of “Six Climatic Factors” into “Fire-pathogen” requires “Shaoyang”.The treatment is based on the principle of “strengthening the body and clearing the evil”,so chooses the classic ancient prescription Qinghaobiejiatang (QHBJT).UseArtemisiae annuae herbato treat Shaoyang Channel,andTrionycis carapaxtreat Jueyin Channel.Moutan cortexandAnemarrhenae rhizomaclear the heat inside,andRehmanniae radixnourish Yin,so that the “Evil Qi” is distributed from the “Blood Aspect” to the “Qi Aspect”,from the inside to the outside.

However,the components of QHBJT are more complex,each component corresponds to many targets,and the targets can have synergistic or antagonistic effects.It is difficult to clarify the mechanism of the prescription for the treatment of renal interstitial fibrosis.The network pharmacology is based on various biological information databases,searching for chemical components and protein targets of the compound to be studied,and also including the record of the disease target.Through the network analysis of the drug and disease,it is helpful to explain the compound treatment of the disease.The mechanism of action,and network pharmacology emphasizes multi-target pathway research,which is consistent with the overall concept of Chinese medicine 8.

Materials and methods

Screening of related targets of QHBJT

Through TCMSP (https://tcmspw.com/tcmsp.php) and TCMID (http://www.megabionet.org/tcmid/) to find the chemical composition of 5 Chinese medicines.After completion,search for the target corresponding to the candidate compound through TCMSP,and use the Uniprot database (https://www.uniprot.org) to correct the protein target of the compound to the standard gene name.

Screening of related targets for renal interstitial fibrosis

Using “renal interstitial fibrosis” as the key word,search the GeneCards database(https://www.genecards.org) to get related targets of renal interstitial fibrosis.

Acquisition of common targets of QHBJT and renal interstitial fibrosis

With the aid of R software,the predicted targets of QHBJT and the targets of diseases related to renal interstitial fibrosis were crossed to obtain a common target,which was used as a candidate target of QHBJT for the treatment of renal interstitial fibrosis.

Construction of the control network of QHBJT

The correlation summary and target prediction results between the active ingredients of the drug and the disease-related gene targets analyzed in 1.2 are used to construct a drug-component-target-disease network model using Cytoscape 3.7.2.The different “nodes” in the figure (node) respectively represent QHBJT,active ingredients of the drug,target and renal interstitial fibrosis,and “edge” represents the relationship between the two.Use Cytoscape’s built-in Network Analyzer tool to analyze the network,focusing on the degree value of the node,that is,the size of the node.The larger the node,the greater the degree value,the more biological functions involved,and the higher the importance.This is to study the more important components and targets in QHBJT and the relationship between them.

Composition of QHBJT-PPI network construction of targets related to renal interstitial fibrosis

In order to further explore the interaction between candidate target proteins,the common target was entered into the STRING database (Version 11.0,https://string-db.org),and a protein-protein interaction network model (protein-protein interaction,PPI) was constructed.Set the biological type to “Homo sapiens”,the minimum interaction threshold is set to “highest confidence” (>0.4),and the rest of the settings are the default settings,hide the points that are not connected to other nodes,and get the PPI network.Import the obtained data into cytoscape and calculate the degree,and finally take the points greater than the median degree as the core target to draw the core target PPI.

Composition of QHBJT-common target and pathway enrichment analysis of renal interstitial fibrosis

Use R software to perform Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)analysis of candidate targets.GO includes three parts:molecular function (MF),biological process (BP) and cellular component (CC).It is used to interpret the biological processes of key targets;KEGG is used to study the main signal pathways involved in key targets.Based on the related targets mapped by the KEGG results,a “common target-pathway” network diagram was constructed to further screen the key target genes of QHBJT in the treatment of renal interstitial fibrosis.

Results

Obtaining the target of the active ingredient of QHBJT

From the TCMSP and TCMID databases,126 chemical constituents ofArtemisiae annuae herba,16 chemical constituents ofTrionycis carapax,49 chemical constituents ofRehmanniae radix,81 chemical constituents ofAnemarrhenae rhizoma,and 55 chemical constituents ofMoutan cortexwere preliminarily extracted.After deduplication of all components,317 were left.The drug target was converted into a gene symbol in uniport,and the duplicate value was checked again,and the 837 repeat value was removed,leaving 166 traditional Chinese medicine targets.

Acquisition of targets related to renal interstitial fibrosis and acquisition of targets shared by drugs and disease targets

2,737 renal interstitial fibrosis-related targets were obtained from the Genecards database,and the drug and disease targets were crossed through Venny 2.1.0,and 102 common targets were obtained,as shown in Figure 1.

Construction of “drug-component-target-disease” network

With the help of Cytoscape 3.7.2,the above-mentioned potential active ingredients and targets are used to construct a network of “active ingredients of QHBJT-common targets”,as shown in Figure 2.The network consists of 241 nodes and 743 edges,and the size of the node represents the corresponding degree value.Use the Network Analyzer in Cytoscape to analyze the node degree value,and the compounds with degree value >5 are shown in Table 1.Among them,the top 4 are quercetin,apigenin,kaempferol,and luteolin,corresponding to 60,30,25,and 24,respectively.These may be the more important active ingredient of QHBJT in the treatment of renal interstitial fibrosis.

Table 1 Active ingredients of QHBJT (degree value >5)

Table 1 Active ingredients of QHBJT (degree value >5) (continued)

Table 2 Key targets contained in critical pathways

Composition of QHBJT-PPI network construction of common targets of renal interstitial fibrosis

The 102 common targets of drugs and diseases were submitted to the STRING11.0 platform,and the PPI network of components of QHBJT-related targets of renal interstitial fibrosis was obtained,as shown in Figure 3.The PPI network contains 102 targets and 2,792 edges.In order to further study the function of each target,the data obtained from the STRING platform is imported into Cytoscape,the degree is calculated.The point whose degree is greater than the median is taken as the core targets,There are 41 core targets.Draw the core targets point PPI,as shown in Figure 4.The gradual change of the node area from large to small,and the gradual change of color from warm color to cool color represents the change of degree value from large to small.Therefore,the larger the area of the node and the redder the color,the greater the degree value of the node,which means the more important position of the node in the network.Among them,INS,TP53,IL6,VEGFA,MAPK8,EGFR,CASP3,MYC,ESR1,EGF have many interaction relationships.

Figure 1 Venn diagram of components of QHBJT-related targets of renal interstitial fibrosis.QHBJT,the classic ancient prescription Qinghaobiejiatang.

Figure 2 “QHBJT active ingredients-common targets” network.QHBJT,the classic ancient prescription Qinghaobiejiatang.

Enrichment analysis of target function and pathway

Use R software to perform GO enrichment analysis on 41 core targets.The results showed that a total of 1,743 GO terms showed enrichment,of which 1,657 were biological processes,21 were cell composition,and 65 were molecular functions.According to thePvalue,the first 12 were selected and drawn into a GO enrichment analysis bar chart using R language.The ordinate of the bar graph represents the three basic categories of GO,BP,CC,MF,and various lower-level items,and the abscissa represents the number of genes annotated to a certain item.The longer the bar graph,the more annotated genes,and the color from red to blue represents thePvalue from small to large,as shown in Figure 5A.It can be seen from the results that QHBJT acts on renal interstitial fibrosis by regulating various BP (P<0.01),of which the top 5 are the response to steroid hormones,cellular response to oxidative stress,regulation of apoptotic signaling pathway,response to oxidative stress,response to radiation.According to the results of CC analysis,transcription factor complex,membrane raft,membrane microdomain,membrane region,and nuclear chromatin are the main sites of action.The first 5 MF include DNA binding transcription activator activity,RNA polymerase II specificity,RNA polymerase II transcription factor binding,ubiquitin protein ligase binding,and nuclear receptor activity.

Figure 3 Components of QHBJT-PPI network of renal interstitial fibrosis-related targets.QHBJT,the classic ancient prescription Qinghaobiejiatang.

Figure 4 Components of QHBJT-PPI network of core targets related to renal interstitial fibrosis.QHBJT,the classic ancient prescription Qinghaobiejiatang.

Use R language to perform KEGG pathway enrichment analysis on the 41 targets obtained,and obtain 131 signal transduction pathways.The first 12 signal pathways with the most enriched targets are drawn by R program to draw KEGG pathway enrichment analysis bubble chart.The left side is the top-ranked enrichment name;the color of the bubble from red to blue represents thePvalue from small to large.The larger the bubble,the greater the gene count of the pathway.The horizontal axis represents the total input genes of the pathway.The ratio is shown in Figure 5B.From the results,it can be seen that the main related pathways involved in the treatment of renal interstitial fibrosis by QHBJT are Kaposi’s sarcoma-associated herpes virus infection,colorectal cancer,prostate cancer,human cytomegalovirus infection,pancreatic cancer,colorectal cancer,measles,PK3K-Akt signaling pathway,apoptosis,fluid shear stress and atherosclerosis.

In order to further reveal the relationship between signal pathways and candidate targets,a “pathway-target” network was constructed,as shown in Figure 6.It can be seen from the network that FOS,IL6,MTOR,MAPK8,RELA,CCND1,TP53,EGFR,and CASP3 have larger degrees and are key targets,which are consistent with the key targets of the PPI core network above.

Figure 5 GO enrichment analysis bar graph (A) and KEGG enrichment analysis bubble chart (B).GO,Gene ontology;KEGG,Kyoto Encyclopedia of Genes and Genomes.

Figure 6 “Pathway-target” network diagram

Discussion

Potential active ingredients

The “drug-compound-target-disease” network predicts that quercetin,apigenin,kaempferol,and luteolin may be the key active ingredients.Quercetin inMoutan cortexandArtemisiae annuae herbais a natural flavonoid compound with biological activities such as anti-tumor,anti-inflammatory,anti-oxidant,anti-platelet aggregation and scavenging free radicals 9.Studies have found that quercetin can inhibit M1 macrophage signal transmission through nuclear factor kappaB (NF-κB) and interferon regulatory factor 5(IRF-5) to reduce kidney damage and inhibit M2 macrophages with cell activation,quercetincanalso reduce the excessive accumulation of extracellular matrix through the TGF-β/Smad pathway 10.The apigenin inArtemisiae annuae herbacan resist kidney damage caused by 3-chloropropane-1,2-diol(3-MCPD) through the caspase cascade pathway 11.Kaempferol,a common component ofArtemisiae annuae herbaandMoutan cortex,can improve acute kidney injury induced by hyperglycemia,reduce oxidative stress,and reduce the expression of inflammatory cytokines (TNF-α and IL-1),fibrotic factors (TGF-α1) and extracellular matrix 12.The luteolin inArtemisiae annuae herbais a flavonoid compound,which inhibits renal artery contraction by reducing the intracellular calcium ion concentration,dilates the already contracted renal artery,lowers blood pressure,and ultimately improves renal function 13.Turtle shell has the effect of inhibiting the hyperplasia of connective tissue,increasing plasma protein,regulating the body’s immune function,and prolonging the existence of antibodies.The anti-kidney fibrosis and kidney protection effects of the classic ancient prescription Biejia Jianwan and the Chinese patent medicine Fufang Biejia Ruangan Tablet are equivalent to ACEI/ARB drugs 14.Shengdi has anti-inflammatory and anti-oxidant properties,lowering blood sugar,improving autonomic nervous system and cognitive functions,anti-depression and immunomodulatory effects.It is used to treat various inflammatory diseases and metabolic diseases,especially for cervical cancer,renal hypertension,and liver damage 15.Studies have found that Qinghaobiejia decoction can adjust the Th1/Th2 ratio in peripheral blood T lymphocytes of patients with systemic lupus erythematosus,and make it more balanced 16.In addition,modern pharmacological studies of this prescription show that it has significant therapeutic effects on immune disorders such as tumors,blood diseases,and chronic wasting diseases,and it can significantly improve the patient’s immune level and accelerate disease recovery 17.

Key gene targets

The key targets of QHBJT in intervention of renal interstitial fibrosis are FOS,MTOR,MAPK8,TP53,IL6,MAPK8,RELA,EGFR,CCND1,and CASP3.These targets are closely related to renal interstitial fibrosis mechanisms such as increased extracellular matrix,inflammatory response,cell cycle arrest,and microvascular thinning.

The proto-oncogene FOS is a target gene in the form of a dimer in the activating protein 1,and c-fos is the product of theFOSgene,which participates in the biological processes of cell proliferation,differentiation,and apoptosis.TGF-β1 is a group of pro-fibrotic cell growth factors,which play a central role in fibrogenesis 18.Studies have confirmed that TGF-β1 can promote the expression of the transcription factor c-fos,and the gene promoters of some extracellular matrix components also contain activating protein 1 binding sequences,suggesting that the expression of c-fos is directly related to the increase in extracellular matrix expression 19.The mammalian target of rapamycin (mTOR) participates in the formation of 2 complexes with different structures and functions,namely mTORC1 and mTORC2.Studies have found that mTORC2 signaling mediates TGFβ1-induced fibroblast activation,leading to renal fibrosis 20.In the chronic course of glomerulosclerosis model mice,low-dose mTOR inhibitor rapamycin treatment reduces proteinuria,systolic blood pressure,inhibits TGF-β overexpression,collagen I deposition,cell proliferation,and tubular interstitial fibrosis,and then delay the progression of renal function 21.The p38MAPK signal transduction pathway is stimulated by inflammatory factors to rapidly phosphorylate p38,its expression is up-regulated,and its downstream factor NF-κB is activated to induce pro-inflammatory molecules Such as the transcription of TNF-α and IL-6.In addition,TGF-β1 mediated TGF-β1/p38MAPK pathway plays an important role in promoting renal fibrosis 22.The cellular tumor antigen P53 is a key cofactor that regulates the transcription of pre-fibrotic genes initiated by TGF-β1,and TGF-β1 stimulates p53 phosphorylation,promotes the interaction with activated SMAD,and then combines with p53/SMAD3 to form a target promoter.participate in the RIF process together 23.

IL-6 is an important inflammatory factor during renal fibrosis.Blocking IL-6 signal can reduce the level of inflammation in kidney tissues,reduce immune cell infiltration and fibrotic cytokine expression,and reduce the accumulation of fibroblasts 24.The sign of NF-κB activation is the nuclear translocation of the dimeric Rel protein transcription factor.The RELA gene is involved in the regulation of inflammation,immunity,cell apoptosis,cell proliferation and differentiation,and NF-κB plays a key role in the progression of chronic kidney inflammation 25.Studies have found that activating the NF-βB subunit c-Rel in damaged tissues stimulates fibrosis,and deleting c-Rel reduces the fibrosis of multiple organs 26.

Evidence from animal studies indicates that EGFR signaling is also related to the occurrence and development of renal fibrosis.After chronic kidney injury,hypobaric hypoxia is activated,which induces the continuous expression and activation of EGFR,and subsequently causes epithelial cells to stagnate in the G2/M phase of the cell cycle.These cells have the ability to produce large amounts of TGF-β1 and other fibrotic cytokines,leading to renal interstitial fibrosis 27.CCND1 is the regulatory component of the cyclin D1-CDK4 complex,which regulates the cell cycle during the G1/S transition.Studies have found that the autophagy inhibitory effect of hydroxychloroquine makes thymic epithelial cells (TECs) sensitive to growth arrest and extracellular matrix production induced by TGF-β in vitro,mainly by arresting cells in the G1 phase.Maladaptive repair occurs when the damage is severe or persistent and the TECs cannot pass the cell cycle and complete cell division.Therefore,there is a correlation between the increase in the proportion of TECs with cell cycle arrest and the continued progression of kidney damage and fibrosis 28.

Caspase-3 (CASP3) is involved in the activation cascade of apoptosis.A study used CASP3-deficient mice to induce ischemia-reperfusion injury and found that renal tubular injury and renal insufficiency occurred in the early induction period,but no obvious microvascular injury was seen at all stages.From a long-term perspective,CASP3-deficient mice showed slight capillary shedding,renal tubule ischemia,and renal interstitial fibrosis,thereby establishing the main role of CASP3 activation in microvascular thinning and renal fibrosis 29.

GO analysis

The GO results showed that steroid hormone response,cell response to oxidative stress,regulation of apoptosis signal pathways,oxidative stress response,and response to radiation are the main biological processes of QHBJT in the treatment of renal interstitial fibrosis.Anabolic-androgenic steroid (such as testosterone,dihydrotestosterone) can cause a mild and reversible increase in serum creatinine and blood urine nitrogen to irreversible chronic kidney disease.By enhancing the renin-angiotensin-aldosterone system,promoting oxidative stress,inducing cell apoptosis,inducing overexpression of inflammatory cytokines and promoting fibrosis 30.Vitamin D and its metabolites play an important role in calcium homeostasis,bone remodeling,hormone secretion,cell proliferation and differentiation.1,25-Dihydroxyvitamin D3 binds to steroid hormone receptors and induces the expression of hepatocyte growth factor to inhibit the activation of renal interstitial fibroblasts 31.The proximal tubule is also the active site for the synthesis of vitamin D through the action of 1α hydroxylase.Therefore,renal tubular atrophy may lead to vitamin D deficiency,and vitamin D receptor signaling defects will promote renal tubular atrophy and renal fibrosis,thereby forming a vicious circle 32.

The oxidative stress response is characterized by increased levels of reactive oxygen species (ROS) and or reactive nitrogen species in cells.The continuous increase of ROS levels in cells will lead to oxidation of lipid,DNA and protein.,which leads to cell damage.In kidney disease,mitochondria and NADPH oxidase are the main sources of endogenous ROS.Nrf2 is the main regulator of antioxidant response.Under basal conditions,Nrf2 keeps transcription inactive by binding to its main inhibitor Keap-1.ROS-mediated oxidation leads to a conformational change of Keap-1,thereby reversing the proteasome degradation of Nrf2.In the experimental model,the ablation of Nrf2 can cause lupus-like autoimmune nephritis and exacerbate diabetes-induced oxidative stress,inflammation,and kidney damage.Nrf2 activators such as bardoxolone methyl can improve glomerular sclerosis,interstitial fibrosis and inflammation by reducing NF-κB activation 33.

Apoptosis is a way of programmed cell death,which is essential for the normal development and maintenance of tissue balance in multicellular organisms.The two main apoptosis signaling pathways are the external pathways regulated by apoptosis receptors and the internal mitochondria path.The proteins of the Bcl-2 family are key regulators of the mitochondrial pathway,including members that support apoptosis (such as Bax) and anti-apoptotic members (such as Bcl-2),which can regulate the cleavage of some cellular proteins (such as caspase-3),and inactivate it.Recent studies have found that the expression of Bax in the kidney tissue of CRF rats increases while the expression of down-regulated Bfl-2 decreases.Hydrogen sulfide can inhibit apoptosis and inflammation through ROS/MAPK and NF-κB signaling pathways,thereby improving Chronic renal failure in mice 34.

KEGG analysis

Through KEGG pathway analysis,as shown in Table 2,QHBJT mainly acts on RIF through signal pathways such as virus infection-related,tumor-related,apoptosis,signal transduction,fluid shear stress,and atherosclerosis.disease.Pathways related to viral infection include Kaposi sarcoma-associated herpesvirus,congenital cytomegalovirus,measles virus,Epstein-Barr virus;tumor-related pathways include colorectal cancer,prostate cancer,pancreatic cancer.

Many pathogens regulate the body’s immune response through various effectors,which can lead to continuous infection and tissue damage.Especially for hosts lacking Th1 immune response,secondary or repeated exposure to infectious agents in this immune-regulated environment may exacerbate fibrotic reactions.In an experiment,HHV8 infection of a host lacking Th1 immune cells caused more organ fibrosis 34.The main viral infections known to be directly or indirectly related to renal interstitial fibrosis include Epstein-Barr virus,congenital cytomegalovirus,HHV8,and measles virus.Pathogens mainly activate myofibroblasts and M2 macrophages through the Th2 immune response 36.

It is known that fibrosis is closely related to the occurrence,development,metastasis and treatment of cancer.Fibroblasts activated by the microenvironment formed by various factors secreted by tumors are called cancer-associated fibroblasts (CAF).Compared with steady-state tissue fibroblasts,CAF usually exhibits a higher proliferation rate and induces the production of pro-inflammatory cytokines by activating pro-inflammatory pathways,such as signa STAT3 and NF-κB,ECM deposition and activation of ECM-related enzymes 38.Mesenchymal stem cells are an important source of fibroblasts in the tumor microenvironment.In prostate cancer models,tumor-derived CXCL16 can induce mesenchymal stem cells recruitment and differentiation into CAF,and then CAF secretes CXCL12,thereby enhancing metastasis 39.In addition,cancer cells can produce mitogenic and fibrotic factors,thereby promoting the activation of pancreatic stellate cells 40.Among them,pancreatic cancer is one of the diseases most closely related to tumor-related fibrosis.It is estimated that about 60–70% of tumor tissues are composed of proliferative stroma,which is characterized by extensive extracellular collagen deposition and activated CAF 41.In addition,many evidences show that epithelial-mesenchymal transition occurs during the formation of colorectal cancer and tumor invasion,and the latter is involved in the process of chronic inflammation-related fibrosis and mucosal repair 43.

The PI3K/Akt signaling pathway is widely present in eukaryotic cells and plays an important role in cell proliferation,differentiation,and apoptosis 42.In previous studies,PI3K inhibitors can reduce the proliferation and accumulation of fibroblasts after UUO.In addition,the down-regulation of the PI3K/Akt signaling pathway not only inhibits expression,but also reduces the number of pro-fibrotic stromal cells 44.

Apoptosis is closely related to renal interstitial fibrosis.Fibrosis-promoting factors represented by TGF-β1 have a pro-apoptotic effect on renal tubular epithelial cells,which makes the latter transitional apoptosis and proliferation decreased,and accelerates interstitial fibrosis.In addition,the activation and proliferation of renal interstitial fibroblasts can secrete a large amount of α-smooth muscle actin to the renal interstitium to promote renal interstitial fibrosis,so the promotion of myofibroblast apoptosis and anti-fibrosis play an important role 46.

Fluid shear stress and activation of the atherosclerotic pathway are important factors in the progression of atherosclerosis 45,and high-cholesterol diet is the main factor in the formation of atherosclerosis.A study has shown that diet-induced hypercholesterolemia Rats can develop renal interstitial fibrosis within a few weeks.The characteristic change of interstitial is lipid accumulation,which causes interstitial infiltration of monocytes.Renal cortex mRNA levels detected an up-regulation of metalloproteinase 1 (TIMP-1) and down-regulation of urokinase and urokinase-type plasminogen activator (μPA),which in turn caused an increase in the matrix in the renal interstitium 47.

In summary,there are multiple effective compounds,pathways of action,and target genes in the treatment of RIF by QHBJT,which embodies the overall concept of disease prevention and treatment of Chinese medicine,and provides new ideas for the treatment of RIF,and also for more Further understanding of the mechanism of action of Chinese herbal compound in the treatment of RIF disease provides a new reference.

Conclusion

According to the theory of “Fuxie” and the differentiation of symptoms and signs of the 6 Channels,we believe that renal interstitial fibrosis is due to “Insufficiency of Right Qi” and the inability to prevent “Evil Qi” from going out.The “Evil Qi” is lurking in “Construction-blood”.With the seasons,external feelings,recovery,physical weakness and other factors,the disease recurs.Local or systemic inflammatory reaction occurs during the attack,but the right and the evil cannot compete with each other.The disease becomes chronic.The “Evil Qi” lurks in the “Triple Yin”,“Phlegm” and “Stasis” are formed over time.And the toxins are added to endogenous,intertwined into a tangible disease nest,and renal interstitial fibers tissue hyperplasia is the same,and “Fuxie” causes fatigue,which is the same as renal failure.We seize the basic pathogenesis of renal interstitial fibrosis as “Healthy Energy Deficiency and Evil Excess”.The disease is located in the “Lesser yang” and “Reverting Yin”.Use the “Five Methods and Six Classics” of “Torship Method”,“Cleaning Method”,“Activating Blood,Resolving Phlegm,and Detoxification Method” 7,using QHBJT to transfer “Fuxie” from “Lesser yang”.

In this study,the method of network pharmacology was used to explore the effective components,targets and related pathways of QHBJT in the treatment of renal interstitial fibrosis,which can provide reference for in-depth study of the mechanism of action in the later period.Although the systematic and holistic thinking of network pharmacology and the characteristics of multi-component,multi-target,and multi-pathway of traditional Chinese medicine have the same goal,it is still unable to fully reveal the characteristics of the component-target-pathway,and the effective components of this study The prediction is based on the existing database,so the results of the study need to be verified in vivo and in vitro.