The exploration of active components of 701 Dieda Zhentong patch and analgesic properties on chronic constriction injury rats

doi:10.21203/rs.3.rs-4807895/v1

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The exploration of active components of 701 Dieda Zhentong patch and analgesic properties on chronic constriction injury rats

https://doi.org/10.21203/rs.3.rs-4807895/v1

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An increasing number of traditional Chinese medicine(TCM) have been confirmed to possess analgesic bioactivity. 701 Dieda Zhentong patch(701-DZP) which includes 14 kinds of TCMs exhibited excellent efficacy in alleviating back or leg pain after a soft-tissue injury. In this study, UPLC/MS was used to construct the fingerprint and potential bioactive ingredients of 701-DZP. 21 compounds were detected and identified in the fingerprint including 12 compounds that pass through the skin and 6 compounds observed in the plasma. Then, the role of 701-DZP in neuropathic pain(NPP) was assessed by network pharmacology and CCI rats. 701-DZP inhibited pain sensitization(MWT and TWL) and the release of inflammation mediators(IL-1β and IL-6) in CCI rats which were in keeping with the core targets of the PPI network. The results of IHC and Western blot showed that the expression of the P2X3 receptor in the DRG and SC of CCI rats was significantly reduced after the treatment with 701-DZP. Moreover, the 701-DZP down-regulated the level of phosphorylation of ERK1/2 MAPK instead of P38 MAPK in the DRG of CCI rats. In conclusion, this study has clarified 6 potential analgesic active compounds of 701-DZP and explored the analgesic properties, which may inhibit the expression of the P2X3 receptor to reduce the release of inflammatory mediators based on the ERK1/2 MAPK pathway to alleviate the NPP.

neuropathic pain

P2X3 receptor

CCI rats

traditional Chinese medicine

701-DZP

Neuropathic pain(NPP) is a chronic pain usually caused by painful neuropathies, nerve injury, herpes zoster, and SC injury^[1]. At present, 10% of the general population suffers from NPP, which is characterized by spontaneous pain, hyperalgesia, and allodynia^{[2, 3]}. Many patients show low response and resistance to existing treatments of NPP and first-line drugs such as gabapentin and pregabalin exhibit various side effects^[4–6]. NPP imposes a severe mental and financial burden on patients, which increases the requirements for novel treatments.

In the last decades, more and more evidence shows that plastic alterations of the nerve based on peripheral or central nerve injury are an important mechanism of NPP. Changes in the balance of excitatory(glutamatergic enhanced) and inhibitory(GABAergic and glycinergic reduced) synaptic transmission in the SDH lead to the excitation of brain projection neurons is the basis of NPP^[7–9]. In addition, the activation and sensitization of ion channels and pain-related targets occur in the condition that action potentials are generated by nociceptive information in the distal ends of the C-fibers or Aδ-fibers of primary afferent neurons^{[10, 11]}. Purinergic P2X3 receptors, receptors expressed on the neurons of the dorsal root ganglia (DRG) and SC, play crucial roles in modulating NPP in various models^[12]. Studies showed that the release of inflammatory mediators in neurons may associated with the activation of the P2X3 receptor in the NPP state. Inflammatory mediators such as IL-1β, TNF-α, and IL-6 are important factors in neuronal sensitization and maintenance of NPP^{[13, 14]}. The inhibition of the P2X3 receptor in the SC alleviates thermal and mechanical hyperalgesia in chronic constriction injury(CCI) rats, the possible mechanism is reduced extracellular regulated protein kinases 1/2 (ERK1/2) phosphorylation^{[15, 16]}.

In general, the complexity of TCM ingredients and the diversity of targets limit its application. Therefore, it is very crucial to elucidate the active ingredients and mechanism of TCM. 701-DZP is a listed patch of Guangzhou Baiyunshan Pharmaceutical Group Co., LTD. Baiyunshan Hejigong Pharmaceutical Factory. It consists of a variety of traditional Chinese medicines including Eupolyphaga Steleophaga, Aconiti Kusnezoffii Radix, Strychnos nux-vomica L, Rhubarb, Zanthoxylum nitidum(Roxb.)DC, Scutellaria baicalensis Georgi, Phellodendron chinense Schneid, Lignum Dalbergiae odoriferae, Polygonum cuspidatum, Borneol, Camphor, Peppermint oil, and Methyl salicylate. A variety of TCM in 701-DZP such as Strychnos nux-vomica L^[17], Scutellaria baicalensis Georgi^{[18, 19]}, and Aconiti Kusnezoffii Radix^[20] have been shown to have analgesic effects. 701-DZP is usually used to alleviate back or leg pain after a soft-tissue injury. However, the analgesic ingredients of 701-DZP and its effect on NPP are still unclear.

In this study, the fingerprint of 701-DZP was constructed and potential active components were identified which could permeate the skin to plasma. The network pharmacology and CCI rats were used to excavate the analgesic properties and mechanisms of 701-DZP. The results suggested that in the constructed fingerprints of 701-DZP, 12 compounds can permeate the skin, and 6 compounds end up detected in the plasma. The release of inflammatory mediators and the counts of inflammatory cells in the DRG and SC were reduced after the treatment of 701-DZP as predicted by network pharmacology. It is analgesic effect may depend on decreased phosphorylation of ERK1/2 by regulating the P2X3 receptor.

2.1 Reagents

LC-MS grade acetonitrile was purchased from MREDA. Formic acid (LC-MS grade) was purchased from Fisher Scientific. Methanol(LC-MS grade) was purchased from Macklin Inc. Water was purchased from A.S.WATSON TM LIMITE.

2.2 The construction of the fingerprint of 701 DZP

2.2.1 UPLC parameters

The separation was performed on both Q Exactive plus UPLC (Thermo Scientific, Germany) and Agilent 1260 UPLC (Agilent, China) with an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm). The flow rate was 0.3 ml/min and the column temperature was maintained at 30°C. The mobile phase consists of acetonitrile(A) and 0.2% formic acid water (B). The gradient elution condition was set as: 0–6.0 min, 15%→24% B;6.0–16.0 min, 24%→45% B; 16.0–19.0 min, 45%→51% B;19.0–20.0 min, 51%→80% B; 20.0–24.0 min, 80% B. Characteristic chromatographic patterns were obtained using 284 nm as the detection wavelength. The auto-sampler temperature was set at 4 ℃, the injection volume was 8 µL for all samples(701-DZP) corresponding formulations, vitro Permeation and Release samples, and rat plasma samples).

701-DZP was sonicated with 25 mL of 70% (v/v) methanol water for 30 min. Then, the crude extracts were centrifuged at 12,000 rpm for 10 min. Finally, the supernatants were filtered through a 0.45 µm PTFE filter before UPLC-ESI-MS/MS analysis.

2.2.2 HRMS parameters

The MS analysis was performed on Q-Exactive Plus Orbitrap MS (Thermo Scientific, Germany) with a heated electrospray ionization (HESI) source. A high-resolution mass spectrum was acquired at full scan in a mass range of m/z 100–m/z 1,500 at a resolution of 70,000 with an AGC target at 3e6. The dd-MS2 data were obtained at a resolution of 1400 with an AGC target at 1e6. Mass spectrometric detection was performed in positive and negative ion modes. The ion source parameters were set as follows: sheath gas and auxiliary gas of nitrogen (purity ≥ 99.99%) with a flow rate of 45 arb and 10 arb; capillary temperature of 320°C; spray voltage of 3,800/3,500 V (+/-); probe heater temperature of 320°C. The stepped normalized collision energy (NCE)was set at 15, 30, and 45. S-Lens RF Level was 55.00.

2.3 Skin permeation studies

The skin permeation studies were carried out in duplicate(Transdermal matrix, 701-DZP) through rat skin using an automated Logan transdermal diffusion cell sampling system (DHC-6T, Logan Instrument Corporation, Avalon, NJ). The system has a receptor compartment capacity of 5 mL and a diffusion area of 1.77 cm2 membrane for the permeability studies was the dorsal section of full-thickness skin from Sprague-Dawley rats, whose hair had been removed on the previous day with an electric clipper. The freshly excised rat skin samples were washed carefully with saline and Stored at + 4°C short term (1 week). The receiver compartment was filled with 5 mL of 20% ethanol followed by the stirred with a magnetic bar. The receptor compartment was maintained at a constant temperature (37.0 ± 1.0°C). DHC-6T instrument was washed and stabilized for 10.0 min. After stabilization, full-thickness rat skin was sandwiched between the donor and the receiver chamber. The transdermal patches with backing membranes were quickly and firmly pressed onto the rat skin. Once adhesion to the skin surface had been confirmed, 2ml samples were withdrawn from the receptor compartment at predetermined time intervals of 0.5, 1, 2, 3, 4, 6, 8, 12, 16, and 24 h. All the samples were stored at − 80°C until analysis. The samples were thawed on ice, and then the samples were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was added into the detection bottle for fingerprint analysis through the in-house developed and validated UPLC-MS/MS method.

2.4 Active compounds analysis of 701 DZP in rat plasma

The hair on the backside of the rat was removed with an electric hair clipper on the previous day of the experiment. Following an overnight fast, rats were divided into two groups (n = 6). Group I served as control, animals of group I were applied with a 1.7 cm2 transdermal matrix system prepared with Glycerol. Group II was administered with 701-DZP. At time intervals of 2, 4, and 8 h after treatment, blood was collected from orbital sinuses. Plasma was collected after centrifugation at 3,500 rpm for 10 min at 4°C. All the samples were stored at − 80°C until analysis. The serum samples were thawed on ice, and then 100 µL of the sample was added to 400 µL of ice-cold methanol and then vortexed for 30 s. Next, the samples were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was transferred into another 2 mL centrifuge tube and concentrated by water bath for 12 h at 4°C. Every concentrated supernatant sample was mixed with 100 µL of ice-cold methanol and then vortexed for 1 min. Then, the samples were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was filtered through a 0.22 µm membrane and added into the detection bottle for Active compounds of 701-DZP in rat plasma LC-MS detection.

2.5 Network Pharmacology

2.5.1 Data Preparation

The Traditional Chinese Medicine Systems Pharmacology(TCMSP) database (http://lsp.nwu.edu.cn/index.php) and Swiss Target Prediction (http://www.swisstargetprediction.ch/) were used to collect all the active chemical composition and targets of 701-DZP based on the keywords Eupolyphaga Steleophaga, Aconiti Kusnezoffii Radix, Strychni Semen, Rhubarb, Zanthoxylum nitidum(Roxb.)DC, Scutellaria baicalensis Georgi, Phellodendron chinense Schneid, Lignum Dalbergiae odoriferae, Polygonum cuspidatum, Borneol, Camphor, Peppermint oil, and Methyl salicylate. Target names were matched with gene symbols by using the UniProt protein database. Gene symbols after merging and removing the duplications were regarded as the targets of 701-DZP.

Importing the words "neuropathic pain" or " neuralgia" to the GeneGards database(https://www.genecards.org), DisGeNET(https://www.disgenet.org), and OMIM databases (http://www.omim.org) to obtain disease targets by limited the species to Homo sapiens. After removing the duplicated targets, NPP-related targets were obtained.

2.5.2 Construction of PPI Network

The targets of 701-DZP and NPP were uploaded to the tool Venny 2.1.0(https://csbg.cnb.csic.es/BioinfoGP/venny.html) to excavate the intersection targets. Construction of protein-protein interaction (PPI) network by extracting and submitting the intersection targets to the STRING database (https://string-db.org). Limited the organisms as "Homo sapiens" and visualized the PPI network using Cytoscape 3.8.2.

2.5.3 GO and KEGG Enrichment

To clarify the gene function and signaling pathways of intersection targets, Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes database (KEGG) enrichment analysis were conducted by using R package clusterProfiler(version 3.14.3) with the threshold p < 0.05 and FDR < 0.1. The Top 10 cellular components(CC), molecular function(MF), biological process(BP), and signaling pathways were visualized.

2.6 Construction of CCI Rats and Administration

2.6.1 Animal

6–8 weeks and 190-210g SPF female Sprague-Dawley rats were purchased from Zhuhai Baishitong Biological Technology Co., LTD. All the animal experiments were approved by Experimental Animal Ethics Committee of Guangdong Huawei Testing Co., Ltd. (Approval number: 202307005). All rats were located in Jinan University Laboratory Animal Center with a standard temperature of 24°C ± 1°C and a 12 h light/dark cycle. Rats were fed for 1 week to acclimate to the environment(have free access to food and water), then the CCI model was constructed.

2.6.2 CCI Model

All rats were divided randomly into the following groups: Control group, CCI group, Lidocaine patch (1×2cm) group, and 701-DZP (1×1cm, 1×2cm, 1×3cm) groups. The details of CCI surgery as described in the following. Rats were anesthetized with isoflurane to separate the skin and muscle of the right thigh. The right sciatic nerve was exposed and ligated with 4 − 0 silk(repeated 4 times at ~ 1 mm intervals). For the Control group, the sciatic nerve proceeded with the same surgery only without the following ligation.

2.6.3 Administration

The rats in the Lidocaine and 701-DZP group were treated with 701-DZP and Lidocaine patch(1×2cm) and 701-DZP(1×1cm, 1×2cm, 1×3cm) for 7 days after the construction of the CCI model respectively. The 701-DZP or Lidocaine was attached to the right thigh of rats. As for the Control group and CCI group, all rats were applied yellow vaseline which is the accessory of 701-DZP for treatment.

2.7 Behavioral Tests

After the construction of the CCI model for 1, 3, 5, and 7 days, the behavioral assays including the von Fery test and the hot-plate test were used to evaluate the mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL). Before modeling, the MWT and TWL of rats were measured and regarded as the baseline. Rat was allowed to acclimate to the environment for 30 minutes before the von Fery test. The details of the von Fery test and the hot-plate test were described in our previous study^[21].

2.8 HE staining

After the treatment for 7 days, rats were euthanized to extract the L3-L5 segment DRG and SC. These tissues were fixed into the 4% paraformaldehyde solution and paraffin was utilized to embed. Cut the embedded DRG and SC into 5µm thick sections and heal the slices at 60 ℃ for 1 hour. Subsequently, sections were washed with ethanol and immersed in a solution of hematoxylin for 3 min. After rinsing the sections were stained with eosin. Finally, an optical microscope was applied to observe the stained slices.

2.9 Immunohistochemistry

The DRG and SC sections were deparaffinized with xylene for 20 minutes followed by being immersed in a graded series of ethanol solutions(ranging from 100–70%) for 5 min. Antigen retrieval of sections was conducted by incubating with citrate buffer. Subsequently, the sections were incubated in a 3% hydrogen peroxide solution. Washed sections with PBS and incubated with goat serum for 45 min. Following this, the primary antibodies against P2X3 receptors (1:200, Affinity) and Iba-1(1:200, Abcam) were used to incubate with sections overnight at 4°C. Incubated the washed sections with the secondary antibody (goat polyclonal; Abcam; 1: 200) at room temperature. Finally, DAB chromogen solution was added and the sections were observed under an optical microscope.

2.10 Western Blot

L3-L5 segment DRG and SC were dissected from rats and removed into RIPA lysis buffer containing protease and phosphatase inhibitors. Centrifuged at 12,000 rpm for 10 min and remained the liquid supernatant. The concentration of protein was evaluated using a BCA protein assay kit. Added loading buffer to protein samples then heated at 100°C for 10 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was used to separate the protein samples, then transferred to polyvinylidene difluoride(PVDF) membranes. Membranes were blocked with 5% skim milk or BSA for 2h and washed with PBST 3 times. Primary antibodies including P2X3 (1:2000, Affinity), IL-1β (1:2000, Affinity), IL-6 (1:2000, Affinity), GAPDH (1:2000, Affinity), ERK1/2(1:2000, Affinity), pERK1/2 (1:2000, Affinity), P38(1:2000, Affinity), and p-P38(1:2000, Affinity) were used to incubate with menbranes overnight in at 4°C. Membranes were washed with PBST and incubated with the secondary antibody(Proteintech, 1:2000) for 2h at room temperature. The image of the Western blot was observed via a gel imaging system and then processed by ImageJ.

2.11 Statistical Analysis

ImageJ was used to analyze the protein band based on its grayscale. GraphPad Prism 9.0 program was used for statistical analysis and results were shown as means and standard error of means (SEM). Independent-sample t-tests were used to evaluate differences between any given pair of groups. Tukey’s post hoc analysis was conducted to assess differences between any two groups using a one-way analysis of variance (ANOVA). P < 0.05 is considered as a significant difference, and P < 0.01 and P < 0.001 are considered as highly significant differences.

3.1 Fingerprint analysis of 701-DZP

To obtain the most useful chemical information and best separation in the fingerprint chromatograms of 701-DZP, those conditions including the mobile phase compositions, the gradient elution procedure, and the detection wavelength were optimized. The BEH Shield C18 column was found to be more suitable due to its better separation and shapes of peaks. To enhance the resolution, formic acid was added to the binary mixture of acetonitrile water. The resulting optimized mobile phase consisting of acetonitrile and water with 0.1%formic acid solution was chosen for the determination of 701-DZP with a large number of peaks on the chromatogram achieved within 24 min. More detectable peaks could be obtained, and the baseline was well improved at around 284 nm. The optimal UPLC condition used in this study is shown in the UPLC Condition section. The UPLC-UV fingerprints of 701-DZP obtained using the UPLC-DAD are shown in Fig. 1A. Results showed that the 21 target compounds in 701-DZP could be obtained by the UPLC-UV fingerprints.

In this study, we established a rapid UHPLC-ESI-MS/MS method to determine 21 compounds in 701-DZP (Fig. 1) with an MRM mode. The UPLC-ESI-MS base peak chromatograms of 701-DZP are shown in Fig. 1B-C. Compound assignments were made by comparing RT values, and MS data (accurate masses, isotopic distributions, and fragmentation patterns in both ionization modes) of the compounds reported in the literature and the Phytochemical Dictionary of Natural Products Database (Wiley, CRC). Table 2 shows the results of the components Identified on the UPLC-UV fingerprints of 701-DZP. The chromatographic and MS data of these detected 21 target compounds were summarized in Table 1

Table 1

Components Identified in 701-DZP Using UPLC − MS Analysis in Negative and Positive Modes
peak no.	RT (min)	proposed compound	molecular formula	ESI(-)/ Measured (m/z)	ESI(-)/ Expected (m/z)	[M–X]⁻	source	reference
1	2.282	Chlorogenic acid	C₁₆H₁₈O₉	353.09	353.09	[M-H]⁻	Phellodendron chinense Schneid.	1
2	4.102	(3R)-3',8-dihydroxyvestitol	C₁₆H₁₆O₆	303.05	303.09	[M-H]⁻	Dalbergia odorifera T. Chen.	2
3	5.322	d-tetrahydropalmatine	C₂₁H₂₅NO₄	356.19	356.19	[M + H]⁺	Phellodendron chinense Schneid.	1
4	7.978	2-O-Cinnamoyl-1-O-galloyl-β-D-glucoside	C₂₂H₂₂O₁₁	461.11	461.11	[M-H]⁻	Rheum officinale Baill.	3
5	8.592	Physcion 1-O-β-D-glucoside	C₂₁H₁₈O₁₁	445.08	445.12	[M-H]⁻	Rheum officinale Baill.	3
6	9.777	β-D-Glucopyranosiduronic acid	C₂₁H₁₉O₁₁	447.09	446.18	[M-H]⁻	Scutellaria baicalensis Georgi	4
7	10.387	6-methyl-rhein-8-O-β-D-glucopyranoside	C₂₂H₂₀O₁₁	459.09	459.09	[M-H]⁻	Rheum officinale Baill.	3
8	10.738	Emodin-8-glucoside	C₂₁H₂₀O₁₀	431.10	432.11	[M-H]⁻	Polygonum cuspidatum Sieb. et Zucc	5
9	11.042	6-methyl-rhein-8-O-β-D-glucopyranoside	C₂₂H₂₀O₁₁	459.09	459.09	[M-H]⁻	Rheum officinale Baill.	3
10	11.908	Nitidine chloride	C₂₁H₁₈NO₄⁺	348.12	347.12	[M + H]⁺	Zanthorylum nitidum(Roxb.)DC	6
11	12.381	mesaconitine	C₃₃H₄₅NO₁₁	632.31	632. 31	[M + H]⁺	Aconitum kusnezoffii Reichb	7
12	12.67	hypaconitine	C₃₃H₄₅NO₁₀	616.31	616. 31	[M + H]⁺	Aconitum kusnezoffii Reichb	7
13	13.513	Aloe emodin	C₁₅H₁₀O₅	269.04	269.04	[M-H]⁻	Rheum officinale Baill.	3
14	15.462	5,8,2'-Trihydroxy-6,7-dimethoxyflavone	C₁₇H₁₃O₇	329.10	329.07	[M-H]⁻	Scutellaria baicalensis Georgi	4
15	16.142	2′,4′,5-trihydroxy-7-methoxyisoflavone	C₁₆H₁₂O₆	299.09	299.00	[M-H]⁻	Dalbergia odorifera T. Chen	2
16	16.934	Rhein	C₁₅H8O₆	283.02	283.02	[M-H]⁻	Rheum officinale Baill.	3
17	17.095	2′-hydroxyformononetin	C₁₆H₁₂O₅	283.06	283.00	[M-H]⁻	Dalbergia odorifera T. Chen	2
18	17.837	3-hydroxy-5-[(1E)-2-(4-hydroxyphenyl’, henyl)]-benzoic acid	C₁₅H₁₂O₄	255.07	255.28	[M-H]⁻	Rheum officinale Baill.	3
19	19.995	Obacunon	C₂₆H₃₀O₇	455.21	455.21	[M + H]⁺	Phellodendron chinense Schneid.	1
20	20.74	Baicalein	C₁₅H₉O₅	269.04	269.05	[M-H]⁻	Scutellaria baicalensis Georgi	4
21	21.906	Apigenin-6-glucoside-8-arabinoside	C₂₆H₂₈O₁₄	564.33	564.16	[M-H]⁻	Scutellaria baicalensis Georgi	4

3.2 Fingerprint of 701-DZP in vitro permeation samples

The results obtained from the release study are captured in UPLC fingerprints where the peak profile at specific RT represents individual released compounds (Fig. 2). Peak profile of compounds of interest released from 701-DZP and Transdermal matrix using Franz-type cell :(a) Glycerol;(b)0.5 h;(c)1 h;(d)2 h;(e)3 h;(f)4 h;(g)6 h;(h)8 h;(i)12 h;(j)16 h;(k)24 h (Fig. 2). Table 2 displays the total components of cumulative released 701-DZP by the dialysis membrane in time points. The UPLC-ESI-MS peak chromatograms of 11 Permeation Components of 701-DZP are shown in Fig. 3.

Figure 2.(a).The typical UPLC chromatographic profile of Transdermal matrix(Glycerol); (b)~(k).The typical UPLC chromatographic profile of 701-DZP vitro permeation samples(0.5h ~ 24h); (l).The typical UPLC chromatographic profile of 701-DZP sample; (m)~(w).UPLC-ESI-MS base peak chromatograms of a representative 701-DZP Permeation Components with the RT name and numbers of the peaks of the characterized components matching those listed in Table 2.

Table 2

701-DZP Permeation Componentsat Different Time Points (h)
Peak no.	proposed compound	Release Time Points (h)
1	Chlorogenic acid	0.5
2	(3R)-3',8-dihydroxyvestitol	2
3	d-tetrahydropalmatine	0.5
4	2-O-Cinnamoyl-1-O-galloyl-β-D-glucoside	0.5
5	Physcion 1-O-β-D-glucoside	0.5
8	Emodin-8-glucoside	2
10	Nitidine chloride	6
12	hypaconitine	0.5
15	2′,4′,5-trihydroxy-7-methoxyisoflavone	2
16	Rhein	8
17	2′-hydroxyformononetin	2
19	Obacunon	3

3.3 Fingerprint of 701-DZP in vivo permeation samples

To understand the metabolism and mechanism of 701-DZP comprehensively, an efficient and integrated strategy was established based on the UPLC-DAD. When the rat were treated with 701-DZP for 2h, 6 compounds were detected in plasma, including Chlorogenic acid,(3R)-3',8-dihydroxyvestitol, d-tetrahydropalmatine, 2-O-Cinnamoyl-1-O-galloyl-β-D-glucoside, Emodin-8-glucoside and 2′,4′,5-trihydroxy-7-methoxyisoflavone Fig. 3B. Those compounds were accumulated in plasma, and the rats were treated by test formulation for 4h Fig. 3C. However, when the rats were treated with the above drug for 8h, 2′,4′,5-trihydroxy-7-methoxyisoflavone was not detected in plasma Fig. 3D. Furthermore, the rats from the control group were not detected above compounds Fig. 3A. The information on 701-DZP in vivo permeation components is in Table 3.

Table 3

701-DZP in vivo Permeation Components in rat plasma
Peak no.	proposed compound	Release Time Points (h)
1	Chlorogenic acid	2
2	(3R)-3',8-dihydroxyvestitol	2
3	d-tetrahydropalmatine	2
4	2-O-Cinnamoyl-1-O-galloyl-β-D-glucoside	2
8	Emodin-8-glucoside	2
15	2′,4′,5-trihydroxy-7-methoxyisoflavone	2

3.4 Network Pharmacology

Network pharmacology was used to excavate the potential analgesic properties of 701-DZP. As shown in Fig. 4A, 632 targets of 701-DZP and 1998 targets of NPP were retrieved after removing the duplications. 222 intersection targets were found between 701-DZP and NPP then imported to the String to construct the PPI network(Fig. 4B). The core targets of the PPI network which include AKT1, TNF, IL-1β, IL-6, and VEGFA were identified and visualized as shown in Fig. 4C. In addition, a traditional Chinese medicine-ingredients-targets network was constructed to evaluate the potential active components(Fig. 4D). The results of GO and KEGG(Fig. 4E-H) showed that MAPK may be the essential signaling pathway of 701-DZP alleviating the NPP.

3.5 Effect of 701-DZP on MWT and TWL in CCI rat

The results of the von Fery test and hot-plate test are shown in Fig. 5 to evaluate the MWT and TWL of rats on days 1, 3, 5, and 7 after the construction of the CCI model. There was a significant difference between the MWT and TWL in the CCI group and the Control group, indicating the validity of the CCI paradigm. Compared with the CCI group, the MWT and TWL were significantly increased after the treatment with 701-DZP(1×2cm, 1×3cm) and Lidocaine(1×2cm) for 7 days. However, The treatment of 701-DZP(1×1cm) significantly increased the TWL instead of MWT compared with the CCI rat.

3.6 Effects of 701-DZP on inflammation infiltration in DRG and SC of CCI rat

The results of HE staining of L3-L5 segment DRGs are shown in Fig. 6A, an increased quantity of inflammatory cells(neutrophils, lymphocytes, and eosinophils) and apoptotic neurons were observed in the CCI group compared to the Control. However, the treatment with 701-DZP(1×1cm, 1×2cm, 1×3cm) and Lidocaine(1×2cm) inhibited the apoptosis of neurons and decreased the inflammatory cells. Compared to the Control group, vacuolation of neurons and a cluster of glial were observed in the SC of CCI rats(Fig. 6B), and these conditions were reserved in the 701-DZP(1×1cm, 1×2cm, 1×3cm) and Lidocaine(1×2cm) groups.

Fig 6 HE staining of the L3-L5 segment DRG and SC. A. HE staining of the L3-L5 segment DRG. B. HE staining of the SC. Observed under the microscope (×200, ×400).

3.7 Effects of 701-DZP on the expression of inflammatory mediators in DRG and SC of CCI rat

The Western blot showed that the expression of IL-1β and IL-6 in the DRG was significantly increased compared to the Control group(Fig. 7B-D). The treatment with 701-DZP (1×2cm, 1×3cm) for 7 days significantly reduced the expression of IL-1β and IL-6 in CCI rats. In the SC(Fig. 7E-G), the level of IL-1β and IL-6 was up-regulated in the CCI group, and the difference was statistically significant in contrast to the Control group. Compared to the CCI group, the expression of IL-1β and IL-6 in the 701-DZP (1×2cm) group was significantly decreased, however, treatment with 701-DZP (1×3cm) significantly down-regulated the level of IL-6 instead of IL-1β.

The inflammatory cell infiltration and up-regulation of inflammation mediators were observed in the SC, and activation of microglia is associated with inflammation in the central nervous system. Therefore, IHC was used to evaluate the activation of microglia in the L3-L5 SC. As shown in Fig. 7A, the significantly increased expression of Iba-1 was observed in the CCI rat by comparison with the Control group. However, this phenomenon was inhibited by the treatment with 701-DZP (1×1cm, 1×2cm, 1×3cm), and a significant difference was detected. Interestingly, Lidocaine(1×2cm) was also shown to inhibit the activation of microglial.

3.8 Effects of 701-DZP on the expression of P2X3 receptor in DRG of CCI rat

Studies have shown that the activation of the P2X3 receptor regulates the release of inflammatory mediators which causes nerve damage and apoptosis of neurons. Damaged neurons release abundant cytokines that recruit inflammatory cells to worsen inflammation. 701-DZP alleviated the hyperalgesia and inflammation of CCI rats, we further explored the expression of the P2X3 receptor which plays an important role in the NPP. The results of IHC showed that the expression of the P2X3 receptor in the DRG of CCI rats was significantly increased compared with the Control group(Fig. 8A). Obviously, after treatment with 701-DZP(1×1cm, 1×2cm, 1×3cm) and Lidocaine (1×2cm), the up-regulation of the P2X3 receptor was inhibited. Western blot(Fig. 8B-C) showed that the expression of the P2X3 receptor was found a significant difference between the CCI group and the Control. In the 701-DZP(1×1cm, 1×2cm) group and Lidocaine group, the expression of the P2X3 receptor was reduced compared with the CCI group.

Fig 8 Effects of 701-DZP on the expression of P2X3 receptor in the DRG. (A) The expression of the P2X3 receptor in the DRG was evaluated by IHC. (B-C) Effects of 701-DZP on the expression of P2X3 receptor in the DRG, n = 3. Data are indicated as mean ± S.E.M, significant differences are presented as ^#P < 0.05 vs. Control; *P < 0.05, **P < 0.01 vs. CCI group.

3.9 Effects of 701-DZP on the expression of P2X3 receptor in SC of CCI rat

As shown in the results of IHC(Fig. 9A), the significantly increased expression of the P2X3 receptor in the SC was observed in the CCI rats by comparison with the Control. However, this phenomenon was inhibited by the treatment of 701-DZP. Subsequently, Western blot showed that compared with the Control, the expression of the P2X3 receptor in the SC of CCI rats was significantly increased(Fig. 9B-C). After the treatment, the expression of the P2X3 receptor there was a significant difference between the 701-DZP(1×2cm) group and Lidocaine(1×2cm) compared with the CCI group.

Figure 9 Effects of 701-DZP on the expression of P2X3 receptor in the SC. (A) The expression of the P2X3 receptor in the SC was evaluated by IHC. (B-C) Effects of 701-DZP on the expression of P2X3 receptor in the SC, n = 3. Data are indicated as mean ± S.E.M, significant differences are presented as ^#P < 0.05 vs. Control; ***P < 0.001 vs. CCI group.

3.10 Effect of 701-DZP on the phosphorylation of MAPK signaling

Fig 10 Effects of 701-DZP on the phosphorylation of MAPK signaling pathway in the DRG. (B) Effects of 701-DZP on the phosphorylation of the P38 MAPK in the DRG, n = 3. (C) Effects of 701-DZP on the phosphorylation of the ERK1/2 MAPK in the DRG, n = 3. Data are indicated as mean ± S.E.M, significant differences are presented as ^##P < 0.01 vs. Control; **P < 0.01, ***P < 0.001 vs. CCI group.

The result of the KEGG enrichment analysis in the network pharmacology showed that the MAPK signaling pathway may be crucial for 701-DZP alleviating NPP. Compared to the Control group(Fig. 10C), the phosphorylation of ERK1/2 was significantly increased in the DRG of CCI rats. After treatment with 701-DZP, the level of p-ERK1/2 was down-regulated, and the difference was statistically significant in contrast to the CCI group(1×2cm, 1×3cm). However, a significant difference was not found in the phosphorylation of P38.

TCM prescriptions relieve NPP have a long history, especially with the advancement of TCM extraction and analysis technology, various TCM monomers with therapeutic activity have proved to be effective^{[22, 23]}. TCM is characterized by multi-component, multi-target, and multi-mechanism, with the advantage being low adverse effects. Modern pharmacological studies have shown that TCM treats NPP by inhibiting inflammatory response^[24], modulating neuronal excitability^[25], and regulating ion channels^[26]. However, it is very necessary to clarify the active ingredients and targets of TCM for analgesia. In this study, the fingerprint of 701-DZP was constructed by using UPLC/MS and utilized to identify its potential active ingredients. 16 compounds, including chlorogenic acid, tetrahydropalmatine, hypaconitine, and 2′-hydroxyformononetin were shown to pass through the rat skin. Eventually, 6 monomers were detected in the plasma of rats, such as chlorogenic acid and tetrahydropalmatine which have been proven to have analgesic effects.

There is no doubt that the role of inflammation in NPP cannot be ignored. In the condition of NPP, infiltration of immune cells including neutrophils, macrophages, and T cells occurs in the damaged tissue^[27]. Abundant inflammatory mediators such as TNF, IL-1β, and IL-6 were produced by activated immune cells^{[28, 29]}. These inflammatory mediators promote neuroimmune activation and induce pain by binding to the receptors on nociceptors^{[30, 31]}. The sensitization of primary afferent neurons and pain hypersensitivity are also associated with inflammatory mediators. Studies have shown that mechanical and thermal hypersensitivity was observed in the naïve rats after injection of IL-1β^[32–34]. In addition, IL-1β modulates neuronal excitability by affecting various receptors and ion channels such as TRPV1, GABA receptors, and sodium channels. For IL-6, studies have observed similar results that intracerebral injection of exogenous IL-6 induced a decrease in the paw withdrawal threshold and up-regulated the expression of TNF-α and IL-1β^{[14, 35]}. In our study, 701-DZP significantly reduced the release of IL-1β and IL-6 in the DRG and SC of CCI rats. The results of HE staining also showed that 701-DZP reduced the number of inflammatory cells and improved inflammatory infiltration. These results were consistent with the network pharmacology(IL-1β and IL-6 in the core of the PPI network). Interestingly, the activation of microglia in the SC was inhibited after the treatment of 701-DZP. The down-regulation of inflammatory mediators in the SC may be related to the inhibition of microglia. However, the mechanism of 701-DZP inhibited the activation of microglia needs further exploration.

The P2X3 receptor is an ATP-dependent ion channel, which is involved in nociceptive information transmission and sensitizes the central nervous system^{[36, 37]}. The ion channels(Ca²⁺ and Na⁺) on the cell membrane were opened after the activation of the P2X3 receptor, causing loss of cell membrane potential and cell damage^{[12, 38]}. Damaged cells such as microglia and neurons release a variety of cytokines involved in the development of neuroinflammation and pain^[39]. Interestingly, the activation of microglia was inhibited after the treatment of 701-DZP also found in our study. The P2X3 receptor is highly expressed in the DRG and SC in the condition of NPP and is a potential therapeutic target. Studies have shown that the release of inflammatory mediators(TNF-α, IL-1β, and IL-6) is regulated by the P2X3 receptor to mediate the neuronal injury and sensitize primary afferent nociceptors. In general, cells produce biological responses to extracellular signals based on the activation of the ERK1/2 signal by the P2X3 receptor^[40], which is consistent with the results we observed. After the treatment of 701-DZP, the expression of the P2X3 receptor in the DRG and SC was significantly down-regulated compared with CCI rats. In particular, 701-DZP inhibited the phosphorylation of ERK1/2 instead of P38 in the DRG of CCI rats.

In this study, we excavated the potential bioactive monomers of 701-DZP based on the fingerprint of it. Eventually, 6 compounds including Chlorogenic acid, (3R)-3',8-dihydroxyvestitol, d-tetrahydropalmatine, 2-O-Cinnamoyl-1-O-galloyl-β-D-glucoside, Emodin-8-glucoside, 2′,4′,5-trihydroxy-7-methoxy isoflavonewere found in the plasma of rats. However, The analgesic properties of these monomers are still unclear, which needs to be further explored. In subsequent studies, we will try to avoid these limitations, for example, by carefully wrapping the bandage around the administration site. In brief, our study identified the potential bioactive ingredients and explored the effects of 701-DZP on NPP. It was suggested that 701-DZP alleviated NPP by regulating the phosphorylation of ERK1/2 based on the P2X3 receptor further to inhibit the release of IL-1β and IL-6.

In summary, a variety of components of 701-DZP can enter the plasma through the skin of rats, and these components may be the key to its analgesic effect. Network pharmacology and construction of CCI rats demonstrate that 701-DZP may reduce mechanical and thermal hypersensitivity of NPP by inhibiting the release of inflammatory mediators and improving inflammatory infiltration. The mechanism of that may be related to reducing the expression of the P2X3 receptor and the phosphorylation of ERK1/2.

biological process

CCI

Chronic Constriction Injury

cellular component

DRG

dorsal root ganglia

Gene Ontology

ERK1/2

extracellular regulated protein kinases 1/2

IHC

immunohistochemistry

KEGG

Kyoto Encyclopedia of Genes and Genome

LC-MS

Liquid Chromatograph Mass Spectrometer

MWT

mechanical withdrawal threshold

TWL

thermal withdrawal latency

molecular function

MAPK

mitogen-activated protein kinase

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NPP

neuropathic pain

PBS

phosphate-buffered saline

PPI

protein-protein interaction

PVDF

polyvinylidene fluoride

spinal cord

SDH

spinal dorsal horn

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SNI

spared nerve injury

spinal cord

TCM

traditional Chinese medicine

TNF-α

tumor necrosis factor-α

UPLC

Ultra Performance Liquid Chromatography

701-DZP

701 Dieda Zhentong patch

Author’s contribution

Work Jun Meng and Zhenglang Zhang: Conceptualization, Investigation, data curation, Formal analysis, writing-original draft preparation, and Writing-Review and Editing. Yujie Wang: Conceptualization, Investigation. Lina Long: Methodology. Anqi Luo: Investigation. Zhenhui Luo: Investigation. Kexin Cai: Investigation. Hong Nie and Xi Chen: Funding acquisition, Conceptualization, Writing-Reviewing, and Editing. All the authors agreed for the final version to be published. Jun Meng and Zhenglang Zhang contributed equally to this.

Funding declaration

This research was supported by the National Natural Science Foundation of China(No. 8181101216)

Ethics statement

The animal study was reviewed and approved by the Experimental Animal Ethics Committee of Guangdong Huawei Testing Co., Ltd. (Approval number: 202307005).

Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

Data can be made available upon reasonable request.

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The exploration of active components of 701 Dieda Zhentong patch and analgesic properties on chronic constriction injury rats

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Reagents

2.2 The construction of the fingerprint of 701 DZP

2.2.1 UPLC parameters

2.2.2 HRMS parameters

2.3 Skin permeation studies

2.4 Active compounds analysis of 701 DZP in rat plasma

2.5 Network Pharmacology

2.5.1 Data Preparation

2.5.2 Construction of PPI Network

2.5.3 GO and KEGG Enrichment

2.6 Construction of CCI Rats and Administration

2.6.1 Animal

2.6.2 CCI Model

2.6.3 Administration

2.7 Behavioral Tests

2.8 HE staining

2.9 Immunohistochemistry

2.10 Western Blot

2.11 Statistical Analysis

3. Results

3.1 Fingerprint analysis of 701-DZP

3.2 Fingerprint of 701-DZP in vitro permeation samples

3.3 Fingerprint of 701-DZP in vivo permeation samples

3.4 Network Pharmacology

3.5 Effect of 701-DZP on MWT and TWL in CCI rat

3.6 Effects of 701-DZP on inflammation infiltration in DRG and SC of CCI rat

3.8 Effects of 701-DZP on the expression of P2X3 receptor in DRG of CCI rat

3.9 Effects of 701-DZP on the expression of P2X3 receptor in SC of CCI rat

3.10 Effect of 701-DZP on the phosphorylation of MAPK signaling

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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