Mingchen CAO, Lei DONG, Chuan WANG, Wenjing LI, Xinxin WEI, Shasha ZHANG, Hongxia YU, Cheng CHENG, Xue YANG
The Affiliated Hospital of Qingdao University, Qingdao 266000, China
Abstract [Objectives] To observe the clinical analgesic effect of Qianghuo Chushi Decoction (QHCSD) on patients with fasciitis, and explore its possible molecular mechanism based on network pharmacology. [Methods] 120 enrolled patients were randomly divided into experimental group and control group, and were separately treated with QHCSD formula granules and Diclofenac Sodium Enteric-coated Tablets for 4 weeks. The patient’s pain visual analogue scale (VAS) was used as the curative effect indicator. The molecular action mechanism of QHCSD was predicted based on network pharmacology, the active components of QHCSD were screened using TCMSP database, potential targets were predicted by PharmMapper server, compound-target network and protein interaction network were constructed, and GO-based enrichment analysis and KEGG-based biological pathway enrichment analysis were performed. [Results] After treatment, the pain scores in each group were significantly lower than those before treatment (P<0.01), the score of the experimental group was significantly lower than that of the control group (P<0.01), and the total effective rate of the experimental group was 83.33%, which was significantly higher than that of the control group (78.33%, P<0.05). Based on 108 active components in QHCSD, a compound-target network was constructed. The PPI network contained 155 nodes and 527 interaction relationships, and key nodes included FOS, ESR1, NCOA1, RELA, EGFR, MAPK8, IL-6, etc. The GO pathway mainly involved steroid hormone and its receptor activity, RNA polymerase II transcriptional regulator binding, nuclear receptor activity, protein heterodimerization activity and other pathways. KEGG metabolic pathways included PI3K-Akt signaling pathway, Kaposi’s sarcoma-associated herpesvirus (KSHV) infection and other pathways. [Conclusions] QHCSD has a significant analgesic effect on fasciitis, and the PI3K-Akt signaling pathway may be the key pathway for its analgesic effect.
Key words Fasciitis, Qianghuo Chushi Decoction (QHCSD), Network pharmacology, Clinical observation, Analgesic effect
Fasciitis is a kind of non-specific inflammation that occurs in the human body rich in white fibrous tissue beneath the skin. Fasciitis can cause edema, exudation, local microcirculation disturbance and fibrosis. Its clinical manifestations are mainly repeated diffuse pain or soreness in the shoulder and back, tendons, ligaments, joints, palpable indurations or cords, often accompanied by muscle cramps, chills, skin numbness and different degrees of movement disorders, aggravated by fatigue or changes in climate and mood. It is a common clinical chronic pain disease. Traditional Chinese medicine (TCM) has accumulated rich experience in the treatment of fasciitis. Qianghuo Chushi Decoction (QHCSD) has been used clinically in the Affiliated Hospital of Qingdao University for decades. In order to further confirm the application value of the cipher prescription QHCSD in the treatment of fasciitis and explore its possible molecular action mechanism, we selected 120 patients with fasciitis to study its analgesic effect. Using network pharmacology, we explored the possible molecular action mechanism of its analgesic effect, so as to provide a reference for the treatment of fasciitis.
2.1 Clinical observation of the analgesic effect of QHCSD on fasciitis
2.1.1General data. We selected 120 fasciitis patients who were admitted to the Affiliated Hospital of Qingdao University from December 2016 to February 2022, and randomly divided them into experimental group (n=60) and control group (n=60), including 35 males and 25 females in the experimental group, age range from 26 to 74 years old, with an average of (50.34±12.58) years old; 32 males and 28 females in the control group, age range from 25 to 71 years old, with an average of (54.13±16.53) years old. There was no statistical difference in gender, age, duration, attack frequency, disease severity and primary disease between the two groups (P>0.05), and the two groups were comparable.
2.1.2Diagnostic criteria. TCM diagnostic criteria: Based on theTraditionalChineseMedicineSyndromeDiagnosisandEfficacyCriteria, physical examination shows soreness, distension and cold pain in the waist, back and joints; one or more tender points at the lesion site; waist, back and joints are unfavorably turned sideways, the pain has fixed point, and the symptoms progress and repeatedly intensify after rainy days, cold or fatigue; history of chronic lumbar muscle strain; cord-like induration may be palpable in severe cases.
Western medicine diagnostic criteria: based onSurgery, the physical examination shows that the diseased part is mostly the neck and back; local soreness, numbness, dyskinesia or spasm of varying degrees; obvious tenderness in the affected area, muscle tension and cord-like nodules felt; biochemical examination and X-ray show no abnormality.
2.1.3Inclusion criteria: (i) conforming to medical ethics; (ii) meeting the above diagnostic criteria; (iii) age 18 to 80 years old; (iv) onset time≤1 month; (v) no severe cognitive impairment, aware of the research and willing to participate and cooperate ; (vi) good bone marrow, liver and kidney function.
2.1.4Exclusion criteria: (i) acute and severe patients; (ii) patients with malignant tumors, blood system diseases, mental diseases and infectious diseases; (iii) allergic to the drugs in this study; (iv) pregnant or breastfeeding.
2.1.5Suspension and withdrawal criteria: (i) failure to meet the inclusion criteria and irregular records in the case report form; (ii) failure to treat according to the test protocol or to complete the course of treatment, which affects the judgment of efficacy; (iii) failure to complete the treatment due to serious adverse events or personal reasons during the treatment.
2.1.6Treatment protocol. The experimental group was given Atractylodis Rhizoma, Sposhnikoviae Radix, Ligustici Rhizoma Et Radix, Achyranthis Bidentatae Radix, Notopterygii Rhizoma Et Radix, Cimicifugae Rhizoma formula granules, produced by CR Sanjiu Pharmaceutical Co., Ltd., 1 dose per day, 300 mL of boiled water divided into two times and taking in the morning and evening; the control group was given diclofenac sodium enteric-coated tablet 25 mg/time, taking in the morning and evening. Both groups received 1 course of treatment for 7 d, a total of 4 courses of treatment. During the treatment, avoid taking spicy, cold, greasy and irritating foods, avoid combining with other analgesic or anti-inflammatory drugs, observe and make records.
2.1.7Efficacy observation. (i) Efficacy observation indicators. We measured the visual analogue scale (VAS) of pain perception and made records before and after treatment. Based on the visual pain analog scale card developed by the Chinese Medical Association Pain Society, we evaluated the pain degree of the patients. (ii) Efficacy evaluation criteria. In accordance with theTraditionalChineseMedicineSyndromeDiagnosisandEfficacyCriteria, Efficacy index=Difference of pain score before and after treatment/Pain score before treatment×100%. Cured (efficacy index>90%): the original symptoms disappear, the pain in the neck, shoulders and back disappear, the muscle strength return to normal, and people could work and live normally; markedly effective (30% 2.1.8Statistical methods. We conducted statistical analysis with the aid of SPSS 21.0 software, measurement data were expressed by mean and standard deviation, and enumeration data were expressed by number of cases and percentage. For quantitative data such as age, attack frequency, duration, and severity of illness between the experimental group and the control group, which did not conform to the normal distribution, we conducted non-parametric tests; for those conforming to the normal distribution, we conducted thettest. The comparison of two categorical indicators such as gender adoptedχ2test or exact probability method. Efficacy index and effective rate were compared by group design and rank sum test of two samples. All statistical tests were two-sided, andP≤0.05 was considered to be statistically significant. 2.2 Analgesic mechanism of Qianghuo Chushi Decoction based on network pharmacology 2.2.1Research tool. We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)[7]and DrugBank database[8]to obtain active components and their pharmaceutical parameters. Based on the PharmMapper database[9], UniProt[10], Pubchem database[11], we predicted the possible relationship and target information. Using Cytoscape 3.5.1 software, we constructed compound-target interaction network. Based on DAVID database[13], KEGG[14], STRING database[15], and R language 3.5.1[16], we performed the pathway enrichment analysis. 2.2.2Collection and screening of active chemical components. The TCMSP database uses mathematical and computational models to predict the pharmacokinetics of TCM chemical components. We searched TCMSP, and out the active components in QHCSD with reference to the compounds with oral bioavailability (OB)≥20%, drug-likeness (DL)≥0.18 and half life (HL)≥4 h as candidate active components. 2.2.3Prediction of potential targets by molecular reverse docking technology. We saved three-dimensional chemical structure data of QHCSD active components retrieved from Pubchem database in .mol2 format and submitted to PharmMapper to predict relevant targets. 2.2.4Screening of pain-related targets. Using "pain" and "ache" as keywords, we searched the pain-related targets in GeneCards and OMIM databases, and screened the analgesic-related targets of QHCSD active components. 2.2.5Construction of active compound-pain target network. Based on Cytoscape software, we constructed a compound-target network, in which nodes represented compounds and gene targets, connections between nodes represented interactions, and node degree values represented the number of routes connected to nodes in the network. 2.2.6Construction of protein-protein interaction (PPI) network. Through uploading relevant target genes of QHCSD active components to the STRING database, we obtained protein-protein interaction information, and selected interactions with a confidence level of >0.9 and imported them into Cytoscape software to construct a PPI network to explore interactions at the molecular level. 2.2.7GO enrichment analysis. We used David V6.8 database to conduct GO enrichment analysis of Gene Ontology for proteins in the PPI network, to explore the role of target proteins of active components in gene function, to obtain thePvalue of the analysis results, and used R software to plot bar graphs. 2.2.8KEGG pathway enrichment analysis. We performed KEGG biological pathway enrichment analysis on the proteins in the PPI network using the David V6.8 database, and screened the pathways withP<0.01 to determine the reliable pathways, annotated and analyzed, and used R language to plot and analyze the main metabolic pathways. 3.1 Clinical comparison of analgesic effect of Qianghuo Chushi Decoction on fasciitis 3.1.1VAS score comparison. The results are shown in Table 1. There was no significant difference in the VAS score before treatment (P>0.05). After treatment, the VAS score in each group was significantly lower than that before treatment (P<0.01), and the VAS score of the experimental group after treatment was significantly lower than that of the control group (P<0.01). Table 1 VAS score comparison before and after treatment n=60) 3.1.2Clinical efficacy comparison. The results are shown in Table 2. The total effective rate of the experimental group was 83.33%, and the total effective rate of the control group was 78.33%; the difference in the total effective rate between the two groups was statistically significant (P<0.05), and the effective rate of the experimental group was significantly higher than that of the control group. Table 2 Efficacy comparison before and after treatment (n=60) 3.2 Analgesic mechanism of Qianghuo Chushi Decoction 3.2.1Screening of active compounds in Qianghuo Chushi Decoction. According to related parameters, we screened and obtained 108 active compounds, including wogonin, ledebouriellol, patchoulene, neryl butyrate, coniferol, piperitenone, baicalin, kaempferol, palmatine, quercetin, cnidilin, coumarin, bergaptin, nodakenin, norkhelloside, tuberosine A, cimicifugic acid, paeoniflorin, diosmetin,etc. 3.2.2Construction of Qianghuo Chushi Decoction compound-target network. We screened a database of related diseases and obtained 8 801 pain-related targets. Among the 106 related targets of QHCSD active components, 96 targets were related to pain, and the constructed compound-target network contained a total of 346 nodes (Fig.1). Fig.1 Venn diagram of Qianghuo Chushi Decoction target-pain target 3.2.3Protein-protein interaction (PPI) network. We constructed a protein-protein interaction network of QHCSD targets (Fig.2), including 80 nodes and 180 interaction relationships. According to the network topology properties, the key nodes whose betweenness and degree values were greater than the mean (betweenness=0.032 5, degree=8.4) included FOS, ESR1, NCOA1, RELA, EGFR, MAPK8, IL-6, CASP3, VEGFA, AR, ERBB2, PPARG,etc. Fig.2 QHCSD active component-pain target network diagram 3.2.4GO functional enrichment analysis. According to the adjustedPvalue (P-adjust<0.005), we listed top 20 GO pathways (Fig.3), suggesting that the active components of QHCSD mainly involve steroid binding and receptor activity at the cellular and molecular levels, activation of transcription factor binding activity, nuclear receptor activity, heme binding, tetrapyrrole binding, ubiquitin-like protein ligase binding pathways. Fig.3 PPI network 3.2.5KEGG analysis. The results of KEGG channel analysis mainly involved PI3K-Akt signaling pathway, Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, human cytomegalovirus infection, hepatitis B, apoptosis, fluid shear stress and atherosclerosis pathway. According toP-adjust<0.05 (Fig.4), we listed the top 20 pathways and the top 5 pathway enriched gene IDs were shown in Table 3. Fig.4 Bar graph of GO pathway enrichment analysis Table 3 Target gene IDs of KEGG pathway enrichment analysis Fasciitis is a kind of trigger points caused by incomplete treatment of injury and further developed by left local adhesions[4], or sudden drop in temperature causes the blood vessels on the surface of the human body to constrict and expand the deep blood vessels, and the fluid leaks and accumulates; it is a metabolite that stimulates neuroreceptors during muscle spasm or extreme ischemia[5]. In traditional Chinese medicine (TCM), fasciitis belongs to the category of "arthralgia syndrome" and "injury to the muscles of the neck, waist and back"[6-8], and TCM has formed a therapeutic principle of warming the tendons and dredging collaterals, dispelling cold and eliminating dampness, and promoting qi and blood circulation[9]. The efficacy for treating the fasciitis pain is good and reliable[10]. The key proteins of QHCSD, such as GABA, ACHE, CHRM1, C-FOS, ERs,etc., play an important role in the process of pain. ERs are widely distributed in pain transmission and regulation pathways, such as dorsal root ganglia, dorsal horn of spinal cord, spinal nucleus, hypothalamus, and limbic system,etc.[11-14]. For acute nociceptive pain, membrane ERs can interact with endocrine and paracrine estrogens; for secondary chronic pain, nuclear ERs are regulated by interacting with estrogen[11-15]. GABA receptor-gated Cl-channels are a family of channel proteins that mediate physiological processes such as regulation of cell excitability and transmembrane transport. GABAA receptors are widely distributed inhibitory transmitter receptors in the mammalian central nervous system. Drugs acting on GABAA or exogenous GABA can relieve pain[16-18]. Acetylcholine has two types of receptors in the body, muscarinic receptors (G protein-coupled receptors) and nicotinic receptors (ligand-gated ion channel receptors), which are abundant in spinal dorsal horn cells[19-20]and primary afferent neuron cells[21]. Acetylcholine regulates pain through muscarinic receptors and neuronal nicotinic receptors[22]. Postsynaptic M1 receptors at the supraspinal level are involved in analgesia, while presynaptic M2 receptors may modulate pain progression by modulating the release of endogenous acetylcholine[19-22].C-fosis widely present in the central nervous system, and its expression level is low under normal circumstances. Only after the cells of the central nervous system receive exogenous and exogenous stimuli,C-fosactivates transcription, which affects the growth, differentiation, neural plasticity, learning and memory, and damage repair of nerve cells, and also mediates the expression of opioid peptides, enkephalins, nerve growth factor,cck-8 and other genes, and participates in pain regulation.C-fosis not only a marker for stimulation to reach the spinal cord, but also mediates the formation of the basis of emotional changes accompanying pain, systemic arousal responses, and pain memory in the central nervous system[23-26]. The KEGG enrichment results suggest that the PI3K-Akt-mTOR pathway may play a key role in the regulation of pain progression by QHCSD. The PI3K-Akt-mTOR signaling pathway is widely expressed in various cells of the body and participates in various physiological processes such as cell metabolism, proliferation, differentiation, and apoptosis, and is involved in the regulation of pathological pain signals at multiple levels[27-28]. For example, it is involved in synaptic plasticity in the center and may be involved in the formation of nociceptive facilitation after tissue injury in the periphery, and mTOR is widely expressed on the myelinated primary afferent fibers of mouse skin and dorsal root nerves[29]. Inflammation leads to increased expression of Akt and mTOR in the dorsal horn of the rat spinal cord, and is most pronounced in neurons in layers I, III, and IV of the dorsal horn of the spinal cord. Hyperalgesia in model animals is accompanied by the activation of the PI3K-Akt-mTOR signal transduction pathway, which is involved in the regulation of inflammatory pain[30]. The regulation of pain by traditional Chinese medicine compounds is a network-like effect of multiple targets and multiple pathways. It provides a new direction for in-depth study of the molecular mechanism of QHCSD regulating fasciitis pain.
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