Bioinformatic Analysis and Experimental Verification of QJHGD on Caerulein-induced Inflammatory Response in SAP Model Rats Based on TLR4/NF-κB/MyD88 Pathway

Baijun QIN, Xiping TANG, Xin YANG, Xianzhong BU, Wenhao GONG, Yueqiao CHEN, Guozhong CHEN*

1. Guangxi University of Chinese Medicine, Nanning 530001, China; 2. The Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China; 3. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China

Abstract [Objectives] To conduct bioinformatic analysis and experimental verification of Qingjie Huagong Decoction (QJHGD) on caerulein-induced inflammatory response in severe acute pancreatitis (SAP) model rats based on TLR4/NF-κB/MyD88 pathway. [Methods] The effective component groups and potential targets of QJHGD were collected by the network pharmacology method. A drug-component-target network was constructed. The GO and KEGG of targets were enriched and analyzed with the aid of Metascape database, and the target pathway related to SAP inflammation was screened. The SAP rat model was established by caerulein combined with lipopolysaccharide, and QJHGD was intragastrically administered. Pancreatic tissue was observed by HE staining. In addition, enzyme-linked immunosorbent assay and immunohistochemistry were used to verify the anti-inflammatory effect of QJHGD on SAP rats and its regulatory effect on TLR4/NF-κB/MyD88 target pathway. [Results] A total of 105 active components of QJHGD and 148 key targets of SAP were predicted and screened; KEGG was enriched in 320 different pathways including toll-like receptor and NF-κB classical pathways. Animal experiment verified that QJHGD reduced serum amylase, serum lipase activity, IL-6, TNF-α levels in SAP rats; HE staining showed the effect of QJHGD on the pathological changes of pancreas, and QJHGD inhibited the positive expression of key proteins of TLR4, NF-κB and MyD88 in the inflammatory transduction pathway. [Conclusions] The mechanism of QJHGD improving pancreatic injury in SAP rats may be related to down-regulating the expression of key proteins in the TLR4/NF-κB/MyD88 pathway.

Key words TLR4/NF-κB/MyD88 pathway, Severe acute pancreatitis (SAP), Qingjie Huagong Decoction (QJHGD), Inflammatory response, Network pharmacology, Experimental verification

1 Introduction

Severe acute pancreatitis (SAP) is a critical disease resulted from various causes of abnormal activation of trypsin, edema and necrosis of pancreatic tissue, and massive release of inflammatory mediators, which will in turn lead to systemic inflammatory response syndrome and multiple organ dysfunction syndrome. The incidence of SAP in Asian countries including China is about 36-125 for every 100 000 people. It accounts for about 20% of the patients with acute pancreatitis, and the mortality rate can reach 6.5%-26.0%, and the disease progresses rapidly and the prognosis is dangerous[1-3]. The pathogenesis of SAP is very complex. Among various pathogenesis, the "theory of inflammatory mediators and cytokines" play an important role in explaining the pathogenesis and progression of SAP. The extensive enzyme activation in pancreatic acinar cells can promote gland autolysis and cell necrosis, thereby releasing a large number of inflammatory mediators and cytokines into the blood, causing oxygen free radical production and vascular damage, and cascading inflammatory responses aggravate multiple organ damage[4]. TLR4/NF-kB/MyD88 signaling pathway is a key inflammatory transduction pathway and can regulate the expression of various inflammatory mediators and cytokines, and the activation of this pathway plays an important role in the pathogenesis of SAP[5].

Qingjie Huagong Decoction (QJHGD) is a famous in-hospital decoction for the treatment of SAP in the First Affiliated Hospital of Guangxi University of Chinese Medicine. It has been used in the hospital and medical alliance cooperative units for many years, and the clinical effect is remarkable[6]. At present, QJHGD has obtained the national invention patent (No.:ZL201811021893.2). The previous experiment of our team proved that QJHGD can reduce pancreatic hemorrhage, reduce the degree of necrosis, and reduce cell infiltration in SAP rats, but the specific mechanism is not clear. In this study, combined with the analysis of bioinformatics data, we constructed a QJHGD regulatory network and enriched related signaling pathways. The SAP rat model was replicated by caerulein-Lipopolysaccharide (CAE-Lps), revealing that QJHGD plays a role in inhibiting inflammatory response and relieving pancreatitis by regulating the TLR4/NF-κB/MyD88 signaling pathway.

2 Materials and methods

2.1 Network pharmacology prediction of QJHGD

2.1.1Online database and analysis software. Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform (http://tcmspw.com/tcmsp.php) https://www.tcmsp-e.com) and Traditional Chinese Medicine Integrated Database (TCMID) http://www.megabionet.org/tcmid/); drug target database Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN TCM) (http://bionet.ncpsb.org/batman-tcm/), Pubchem (https://pubchem.ncbi.nlm.nih.gov/), Swiss target prediction (http://www.swisstargetprediction.ch/); disease database GeneCards(www.genecards.org/), Therapeutic Target Database (TTD) (http://db.idrblab.net/ttd/), Online Mendelian Inheritance in Man (OMIM) (https://omim.org/), Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB)(www.pharmgkb.org/), and Drugbank (https://go.drugbank.com/). Protein-protein interaction String database (https://string-db.org/) and protein database Uniprot (https://www.uniprot.org). Gene function annotation website Metascape (https://metascape.org/). Drawing tools: Cytoscape 3.7.1, yihanbo website(http://www.ehbio.com/ImageGP/index.php/).

2.1.2Screening of active components and targets of QJHGD. Using TCMSP and TCMID databases, we searched the chemical components of seven traditional Chinese medicines in QJHGD: Bupleuri Radix, Rhei Radix Et Rhizoma, Magnoliae Officinalis Cortex, Scutellariae Radix, Aucklandiae Radix, Persicae Semen, and Aurantii Fructus Immaturus. The screening conditions met the "Lipinski drug-like principle", and the drug-likenessDL≥0.18, oral bioavailabilityOB≥30%, and Caco-2 cell apparent permeability coefficient≥-0.4. We deleted the compounds without literature support by manual search, combined with the results of the reference and PubChem database, removed the compounds that did not meet the pharmacokinetic parameters, and finally retained the active components of QJHGD. The screened active components were combined with Pubchem and Swiss target prediction to pair potential drug targets one by one. Combining the results of the BATMAN TCM database, we obtained a set of QJHGD targets. Gene symbols were annotated through the UniProt database.

2.1.3Screening of SAP related targets. Using "Severe acute pancreatitis" as the key words, we comprehensively collected SAP-related targets from GeneCards, TTD, OMIM, pharmgkb, and Drugbank databases, interactively mapped the QJHGD target set and SAP disease targets, and displayed a set of relevant targets for QJHGD intervention in SAP in the form of an intersection Venn diagram.

2.1.4Construction of protein-protein interaction (PPI) network. We entered the intersection targets into the String website to construct a PPI network diagram. Selected "Muitiple proteins", species "Homo sapiens", set the confidence level to>0.90, the number of kmeans cluster clusters=3, and obtained three types of PPI network diagrams with different colors according to the relationship between the targets.

2.1.5Construction of "drug-component-target" network. The active components of the drug and the target set of QJHGD intervention SAP were mapped to each other, and the component targets were matched one by one. The, we plotted "drug-component-target" network diagram of QJHGD intervention SAP with the aid of Cytoscape 3.7.1 software.

2.1.6GO and KEGG enrichment analysis. Metascape database performed Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for core targets,P<0.05. We selected the top 20 entries with the most enrichment to plot GO and KEGG bubble charts with the aid of the yihanbo website.

2.2Invivoexperimental verification of CAE-Lps-induced SAP model in rats

2.2.1Animals. A total of 40 male SPF SD rats, aged 6-8 weeks, weighing (200±20) g, were purchased from Hunan SJA Laboratory Animal Co., Ltd., license No.:SCXX (Xiang) 2019-0004. Rats were reared in the SPF laboratory of Guangxi University of Chinese Medicine [SYXK (Xiang) 2019-0001] under the following conditions: humidity 55%-65%, temperature (24±2) ℃, and light-dark cycle 12 h/d. All animals involved in the experiments were kept in compliance with welfare standards.

2.2.2Drugs and reagents. QJHGD experimental prescription: Bupleuri Radix, Rhei Radix Et Rhizoma, Magnoliae Officinalis Cortex, Scutellariae Radix, Aucklandiae Radix, Persicae Semen, and Aurantii Fructus Immaturus, prepared by the decoction room of the First Affiliated Hospital of Guangxi University of Chinese Medicine, and the Chinese Medicine Resource Center of Guangxi University of Chinese Medicine identified the medicinal materials as complying with provisions of theChinesePharmacopoeia. The drugs were soaked in 10 times distilled water for 24 h, boiled with strong fire first, and then continued to cook for 30 min with slow fire. The filtrated drug solution was decocted for 3 times using the conventional method, and the filtered drug solution was concentrated with a rotary evaporator to prepare 1 g/mL crude drug. Ulinastatin for injection (specification 2 mL: 100 000 IU, Guangdong Tianpu Biochemical Pharmaceutical Co., Ltd., catalog number 031905103). Elisa reagents: rat α-amylase (α-AMS, Thermo Fisher Scientific DG96286Q-96T), rat lipase (Lipase, Thermo Fisher Scientific DG96276Q-96T), rat interleukin 6 (IL-6, Wuhan Cloud Clone L210405259), rat tumor necrosis factor α (TNF-α, Shanghai Enzyme-linked Biotechnology m1064303-C). Immunohistochemical reagents: hematoxylin-eosin staining kit (Solarbio, catalog number G1120), DAB color development kit (Beijing Zhongshan Golden Bridge Biological Technology, catalog number ZLI-9018), TLR4 primary antibody (Thermo Fisher, catalog number 13-9924-81), NF-κB P65 primary antibody (Thermo Fisher, catalog number PA5-27617), MyD88 primary antibody (Thermo Fisher, catalog number PA5-19918), secondary antibody universal two-step detection kit (rat enhanced polymer method detection system, catalog number PV-9000). caerulein (Shanghai Yuanye Biotechnology, catalog number S62702), lipopolysaccharide (Shanghai Yuanye Biotechnology, catalog number S11060).

2.2.3Instruments. 5804R Centrifuge (Eppendorf, Germany); Multiskan Go Full Wavelength Microplate Reader (Thermo Fisher, USA); ZKPJ-1A Roaster Machine (Tianjin Aihua Medical Equipment, China); Histostar Tissue Embedder (Thermo Fisher, USA); ASP300S Fully Enclosed Tissue Processor (Leica, Germany); CS500 Automatic Glass Coverslipper (Dakewe Biotech, China); Electron Microscope (Leica, Germany).

2.2.4Animal modeling. After one week of adaptive feeding, SD rats were randomly divided into normal group, model group, and TCM group, western medicine group, with 10 rats in each group. Within 24 h before modeling, rats were fasted but provided with water. The modeling adopted the caerulein injection method[7]. The caerulein was dissolved in DMSO and then diluted to 5 mg/L with 0.9% normal saline, with reference toMethodologyofPharmacologicalExperiment[8]. According to the conversion of clinical body weight, the rats in the model group and each administration group were intraperitoneally injected with 50 μg/kg CAE, once every hour, for 6 consecutive times, and the seventh injection of 10 mg/kg LPS was used to replicate the SAP model. After the injection, the TCM group was given QJHGD solution by intragastric administration of 7 g/kg (equivalent to the clinical equivalent dose, and this dose was the best dose determined by the previous experiment, so no dose-effect group was set). The normal group and model group were intragastrically administered with the same volume of normal saline. The western medicine group was given subcutaneous injection of ulinastatin, which was converted to 20 000 IU/kg according to the clinical dose, twice a day for 3 consecutive days. After administration, rats were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital sodium.

2.2.5Effects of QJHGD on serum amylase, serum lipase activity and inflammatory factor expression levels in SAP rats. When collecting blood samples, the abdominal aorta was fully exposed. A trocar was inserted to collect blood, and blood samples were collected. The collected blood samples were placed at room temperature for 2 h, then centrifuged in a high-speed centrifuge at 3 500 rpm for 10 min at 4 ℃, and dispensed the upper serum into test tubes. TheODvalue of serum amylase, serum lipase, IL-6 and TNF-α in each group was detected by enzyme-linked immunosorbent assay microplate reader at the wavelength of 450 nm, and the sample standard curve was plotted and the concentration was converted.

2.2.6Hematoxylin-eosin staining method to observe the effects of QJHGD on the pathological changes of pancreatic tissue in SAP rats. Pancreatic body parts were taken, fixed in 4% paraformaldehyde, then embedded in paraffin, and then baked in a 37 ℃ incubator for 1 h, then dewaxed and hydrated in xylene and anhydrous ethanol. The sections were stained with hematoxylin staining solution for 5 min, and after differentiation, they were soaked in tap water, then placed in eosin staining solution for 1 min, washed with tap water, and finally dehydrated, transparency processed, and mounted for observation of pathological changes of pancreatic tissue of SAP rats in each group including QJHGD group under an electron microscope.

2.2.7Detection of the expression of key proteins of TLR4/NF-κB/MyD88 pathway in pancreatic tissue by immunohistochemical method. After being roasted at 65 ℃, paraffin sections were soaked in sodium citrate repair solution and repaired at high pressure (100 ℃, 100 kPa) for 1 h. After blocking and closing, the antibody was diluted at a ratio of 1∶100 according to the antibody instructions. TLR4, NF-κB, and MyD88 primary antibodies were incubated and stored in a wet box at 4 ℃ overnight. After 24 h, took out PBS and washed 3 times. After incubating for 20 min, removed the secondary antibody and added DAB chromogenic solution, then counterstained with hematoxylin to turn blue, dehydrated, and mounted. We used Image-Pro Plus 6.0 software to analyze and calculate the average optical density. For the calculation method, we referred to the literature of Li Feng[9].

3 Results and analysis

3.1 Network pharmacology prediction of components and targets of QJHGD in the SAP treatment

3.1.1Screening of active components and prediction of targets of QJHGD. We collected a total of 1 415 components of QJHGD were collected in the TCMSP and TCMID databases, and deleted repeated compounds. The components met the drug-likenessDL, oral bioavailabilityOB, Caco-2 cell model conditions and Lipinski drug-like principles. Then, we selected compounds with good ADME properties and deleted components without literature support by manual searches. Pharmacodynamic molecules such as quercetin and hesperidin that meet the conditions of pharmacokinetic parameters in the literature were re-incorporated. Finally, we determined that QJHGD has 105 kinds of active components such as Sainfuran, aloe-emodin, and Magnolol. Specifically, Bupleuri Radix had 13 kinds, Rhei Radix Et Rhizoma had 10 kinds, Magnoliae Officinalis Cortex had 8 kinds, Scutellariae Radix had 32 kinds, Aucklandiae Radix had 5 kinds, Persicae Semen had 18 kinds, and Aurantii Fructus Immaturus had 19 kinds, as shown in Table 1. The screened active components were combined with Pubchem and Swiss target prediction to pair potential drug targets one by one. Combining the results of the BATMAN TCM database, we obtained a total of 189 targets of QJHGD.

Table 1 General information of active components of QJHGD

3.1.2SAP related targets. By searching GeneCards, TTD, OMIM, pharmgkb, and Drugbank databases one by one, the number of SAP-related targets collected was 6 989, 12, 147, 121, and 12, respectively. A total of 7 039 SAP-related target genes were obtained after the combination of the five datasets. The mapping between each dataset is shown in Fig.1.

Fig.1 Venn diagram of SAP target genes in different databases

3.1.3Important targets and PPI networks of QJHGD in the prevention and treatment of SAP. After the intersection of 189 TCM targets and 7 039 SAP related targets intervened by QJHGD, we obtained 167 common targets for the prevention and treatment of SAP, as shown in Fig.2. We entered the common target set on the String website, and after deleting the invalid targets, we obtained 148 important target sets, as shown in Fig.3.

Fig.2 Venn diagram for intersection target genes of QJHGD-SAP

3.1.4"TCM-component-target" regulation network of QJHGD in the treatment of SAP. We input 105 active components of QJHGD and 148 important targets of SAP disease into Cytoscape to plot a "drug-component-target" network diagram, the network contains 318 nodes and 1 167 edges. The diamonds represent disease targets, the circles represent TCM drug components, and the circular nodes of different colors represent different TCM drugs, reflecting the characteristics of QJHGD with multi-component, multi-target,and multi-way prevention and treatment of SAP, as shown in Fig.4. Specifically, TLR4, NF-κB, Myd88 targets were cross-mapped by the active components of TCM drugs such as Bupleuri Radix, Rhei Radix Et Rhizoma, Magnoliae Officinalis Cortex, Scutellariae Radix, Aucklandiae Radix, and Aurantii Fructus Immaturus. Toll-like receptor 4 (TLR4), as a portal initiator protein of inflammatory cascade, is widely distributed in SAP pancreatic acinar cells and intestinal mucosal epithelial cells; nuclear transcription factor kappa B (Nuclear factor kappa B, NF-κB) is considered to be the upstream target of the SAP cascade reaction, and can upregulate the expression of immune-inflammatory-related chemokines and adhesion factors, and promote the continuous amplification of the inflammatory response and aggravate organ damage; Myeloid differentiation factor 88 (MyD88), as an important adaptor protein downstream of the toll pathway, plays a key role in the inflammatory immune pathway. The above three targets may be a pathway by which QJHGD antagonizes the inflammatory response of SAP.

Fig.3 PPI network of QJHGD in the treatment of SAP

Fig.4 Network diagram for "TCM-component-target" of QJHGD

3.1.5GO and KEGG enrichment analysis. The 148 important targets of QJHGD in the prevention and treatment of SAP were input into Metascape for functional and pathway enrichment analysis. Through GO enrichment analysis, we obtained 1 814 biological process entries, 94 cellular component entries, and 172 molecular function entries. Biological processes include lipopolysaccharide reaction, cell reaction to organic cyclic compound and other processes; cell components involve the perinuclear region of the cytoplasm, serine/threonine protein kinase complexes,etc.; molecular functions include transcription factor binding, G protein-coupled amine receptor activation,etc., as shown in Fig.5.

Note: GO-BP: biological processes; GO-CC: cellular components; GO-MF: molecular functions.

Through KEGG pathway enrichment analysis, we obtained a total of 320 different pathways. As shown in Fig.6, the toll-like receptor canonical pathway and the NF-κB pathway were the target pathways of this study. The pathway enrichment analysis also further verified and revealed that QJHGD may play a role in preventing and treating SAP by regulating toll-like receptors and NF-κB classical pathway. The enrichment results are consistent with the important targets of TCM. Subsequent animal experiments were carried out to verify the expression of TLR4/NF-kB/MyD88 pathway proteins.

Note: hsa04620 Toll-like receptor signaling pathway and ko04064 NF-κB signaling pathway are our research objects.

3.2Invivoexperimental verification of animal model

3.2.1Effects of QJHGD on serum amylase and lipase activities and the contents of IL-6 and TNF-α in SAP rats. Compared with the normal group, the activities of amylase and lipase in the serum of the rats in the model group were increased (P<0.05); the levels of both the TCM group and the western medicine group were lower than those of the model group (P<0.05); compared with the western medicine group, the serum amylase activity was lower in the TCM group, and the difference in the expression of serum lipase activity was not significant. The contents of TNF-α and IL-6 in the model group were higher than those in the normal group and each administration group (P<0.05); the content of IL-6 in the TCM group was significantly lower than that in the model group (P<0.05). The above results indicated that QJHGD can effectively reduce the expression levels of serum amylase, lipase, IL-6 and TNF-α in SAP model rats, and inhibit the inflammatory response of rats (Fig.7).

3.2.2Effects of QJHGD on pancreatic histopathology in SAP rats. Histopathological examination of the pancreas of the SAP model rats showed that the structures of the pancreatic lobules and acinars of the rats in the normal group were normal, the tissue structure was meticulous, and there was no obvious edema, hyperemia and inflammatory infiltration; pancreatic acinar atrophy, inflammatory cell infiltration, tissue edema and significant vacuolar-like changes ap-peared in some areas of the model group; most of the pancreatic acinar structure remained in the TCM group and the western medicine group, with only a small amount of necrosis and mild edema (Fig.8).

Note: *denotes P<0.01 compared with the normal group; #denotes P<0.01 compared with the model group.

Note: A. normal group, B. model group, C. QJHGD group, D. western medicine group.

3.2.3Effects of QJHGD on TLR4/NF-kB/MyD88 signaling pathway in the pancreas of SAP rats. In the normal group, the pancreatic tissue structure was intact, and the positive expression of TLR4, NF-κB and MyD88 was still shallow; a large number of cytoplasmic TLR4, NF-κB positive staining cells and nuclear MyD88 positive staining cells were seen in the model group; compared with the normal group, the expressions of TLR4, NF-κB and MyD88 in the model group were all increased (P<0.05), and the positive expression areas were significantly increased. The acinar structure in the TCM group was basically intact, and the positive staining cells for TLR4, NF-κB and MyD88 were significantly reduced compared with the model group and the western medicine group; compared with the western medicine group, the positive expressions of TLR4 and NF-κB in the TCM group were significantly decreased (P<0.05), and the positive expression of MyD88 in the TCM group was not significantly different (P<0.05), as shown in Fig.9-10.

Note: A. normal group, B. model group, C. QJHGD group, D. western medicine group.

4 Conclusions and discussion

4.1 ConclusionsIn this study, we used network pharmacology data analysis, and replicated a rat disease model by combining caerulein and lipopolysaccharide. Throughinvivoexperiment, we found that the mechanism of QJHGD improving pancreatic injury in SAP rats may be related to down-regulating the expression of key proteins in the TLR4/NF-κB/MyD88 pathway, as shown in Fig.11.

Note: *denotes P<0.01 compared with the normal group; #denotes P<0.01 compared with the model group.

Fig.11 Schematic diagram for QJHGD inhibiting inflammatory response mechanism

4.2 DiscussionSAP is a common clinical emergency acute pancreatitis accompanied by persistent organ dysfunction, systemic inflammatory response syndrome, and multiple organ failure. In the early stage of SAP, timely intervention with different treatment methods, such as TCM nasal feeding, enema, external application, is very important to relieve the condition through multiple ways of integrated TCM and western medicine. According to the TCM theory, SAP belongs to dampness-heat and stasis turbidity entangled in the middle energizer and intestines, over time becoming poisonous, consuming qi and damaging yin, so the treatment should combine clearing heat and resolving dampness for detoxification, resolving stasis and preserve yin[10]. QJHGD is a long-term and widely used clinically agreed prescription with proven curative effect in compliance with the above-mentioned treatment principles. It is prepared by seven Chinese medicinal herbs and has functions of removing blood stasis, clearing heat and detoxifying, and can remove poisonous evils, preserve yin and protect yang. QJHGD has definite clinical efficacy, but its target and pathway mechanism are still unclear. This study combined network pharmacology and molecular biology methods to explore the mechanism of QJHGD in the treatment of SAP.

Through the network pharmacology multi-database, we collected the chemical components of QJHGD, and the screening met the requirements of Lipinski drug-like principle and drug likenessDL≥0.18, oral bioavailabilityOB≥30%, and Caco-2 cell apparent permeability coefficient≥-0.4. Combined with PubChem database, we finally retained 105 kinds of active components of QJHGD. The main component of Bupleuri Radix in QJHGD is saikosaponin, which is a natural saponin compound and has the functions of anti-inflammatory, immune regulation, and enzyme secretion. According to findings of Lei Fei[11], saikosaponin can improve the pancreatic exocrine function of rats with pancreatitis by inhibiting the expression of c-fos and c-jun mRNA and protein, thereby inhibiting the excessive synthesis of pancreatic enzymes and proteins and inhibiting the progression of pancreatitis. The main components of Rhei Radix Et Rhizoma are emodin and rhein in anthraquinones, which can regulate blood lipids, resist platelet aggregation, antioxidant and immune regulation. The study of Wu Yang[12]indicates that the high expression of TLR4 promotes the process of inflammatory response, and emodin can restore the Treg/Th17 immune balance by down-regulating the expression of TLR4 and other immune factors, and relieve the inflammatory response of SAP; through injecting sodium taurocholate into the pancreatic bile duct to replicate the SAP animal model, Cai Danli[13]also confirmed that emodin can regulate the level of Toll-like receptor 4 in rat pancreas and lung tissue to exert an anti-inflammatory effect. Magnolol has anti-inflammatory and antibacterial, anti-pathogenic microorganism, anti-ulcer, antioxidant, and anti-tumor effects. The results of Wang Yan[14]indicate that magnolol can inhibit the HMGB1-TLR4/NF-κB signal transduction pathway, reduce the level of inflammatory factors, and relieve SAP and SAP-related lung injury; in addition, magnolol can scavenge oxygen free radicals and improve microcirculation blood perfusion also play an important role in relieving pancreatitis. Baicalin is the main active component of Scutellariae Radix. Ou Jingmin[15]found that baicalin can inhibit the expression of NF-κB in pancreatic tissue and reduce the inflammatory response of the pancreas, and the effect is similar. Aucklandiae Radix contains terpenoids, flavonoids and other components. As one of its active components, costunolide can effectively inhibit lipopolysaccharide-induced NO production and NF-κB activation, and inhibit the expression of inducible nitric oxide synthase (iNOS) gene, to exert the anti-inflammatory effect[16]. Lan Tao[17]found that Persicae Semen extract could significantly reduce the mRNA levels of TLR4 and NF-κB p65 in SAP rats and improve their immune function. The above experiments and literature studies demonstrate the main components of QJHGD play an important role in inhibiting the inflammatory response of pancreatitis and regulating the expression of TLR4/NF-κB p65 pathway.

Through network pharmacology GO and KEGG enrichment analysis, we found that the biological processes of QJHGD preventing and treating SAP include response to inorganic substances, lipopolysaccharide response, and binding to transcription factors. The intersection targets involve classical signaling pathways such as PI3K-Akt, Toll-like receptors, and NF-κB. The occurrence of SAP inflammation is related to the recognition of pathogen-related molecular patterns by pattern recognition receptors (PRRs), the triggering of immune responses, and the release of inflammatory mediators. The Toll-like receptor family is an important inflammatory recognition receptor. This type of transmembrane protein can recognize the extracellular region of receptors and complexes, thereby stimulating downstream molecular pathways. TLR4 is the first type I transmembrane protein receptor to be discovered. The TIR domain of its intracellular domain can combine with the death domain and intermediate domain of the adaptor protein Myd88 to recruit and separate interleukin receptor-related kinases (IRAK), NF-κB inducing kinase (NIK) that activates the downstream pathway after binding to tumor necrosis factor receptor, and activation of NIK further activates the IκB kinase, which by disaggregating with the NF-κB complex exposes nuclear sequences to stimulate the nuclear transcription factor pathway, leading to inflammatory and immune responses[18]. In this animal experiment, we replicated the SAP rat model by intraperitoneal injection of caerulein and lipopolysaccharide. Compared with the retrograde injection of sodium taurocholate into the biliopancreatic duct, this method is easy to operate, has a high modeling success rate, and can stably replicate the SAP model. The experimental results showed that the activities of serum amylase, serum lipase, IL-6 and TNF-α in the model group were significantly higher than those in the normal group. The model group also showed pancreatic acinar atrophy, inflammatory cell infiltration, tissue vacuolar-like lesions, activation of TLR4 pathway, and positive expression of NF-kB P65 and MyD88 proteins in some areas of pancreatic tissue were significantly increased. The expression levels of inflammatory factors such as amylase, lipase, IL-6, TNF-α and other inflammatory factors in the serum of the rats in the TCM group were significantly reduced; the structure of the pancreas was basically intact, there was no obvious edema and hyperemia, the expression of TLR4 was down-regulated, the activation of NF-κB p65 was inhibited, and the positive expression of MyD88 protein was decreased. These demonstrate that QJHGD could inhibit the inflammatory response of SAP rats by down-regulating the TLR4/NF-kB/MyD88 signaling pathway, which was consistent with the target signaling pathway enriched by KEGG in network pharmacology.