Geniposide Alleviates Neuropathic Pain in CCI Rats by Inhibiting the EGFR/PI3K/AKT Pathway And Ca2+ Channels

Neuropathic pain (NP) is a common disorder among individuals worldwide, but there is still no effective treatment for NP. The EGFR pathway promotes NP nociceptive sensitization and represents a potential therapeutic target. Geniposide is abundant in natural plants and has various pharmacological activities, such as analgesia and anti-inflammation properties, which can improve NP, but the specific mechanisms have not been elucidated. The present study first predicted and molecularly docked geniposide targets, suggesting that geniposide may play a role in improving NP by targeting EGFR. This study further clarified that geniposide alleviates NP and improves the inflammatory response using a chronic constriction injury (CCI) model, whereas the administration of an EGFR agonist weakens the above effects of geniposide. Analysis of transcriptome data further suggests that geniposide not only improves CCI symptoms by reducing EGFR/PI3K/AKT pathway activity but also may exert anti-inflammatory effects by inhibiting the Ca2+ signaling pathway. The above results affirm the potential value of geniposide in the treatment of NP and lay the foundation for further clinical application.


Introduction
Neuropathic pain (NP) is a common form of chronic pain caused by somatosensory nervous system lesions or diseases, such as cancer, trauma, and diabetes mellitus, and has a significant impact on the quality of life of patients (Borges et al. 2021). Most patients have persistent or intermittent spontaneous pain, such as burning, tingling, and squeezing sensations, which may be accompanied by evoked pain, especially in response to light touch and cold (Finnerup et al. 2021). There are no effective drugs that clearly target NP; however, considerable research has focused on the molecular mechanisms associated with NP. It is estimated that there are approximately 90 million patients with NP in China.
Among the main current clinical medication treatments, the most common include anticonvulsants, antidepressants, opioid analgesics, and N-methyl-D-aspartate (NMDA) antagonists (Xiao et al. 2021). The current first-line drug for NP, gabapentin (Gab, a Ca 2+ channel regulator), is limited by its multiple side effects (Ghaly et al. 2018;Rayiti et al. 2020). The identification of safer and more effective medications to treat NP is needed.
Studies have shown that growth factors and their cognate receptors promote pain sensitization and represent therapeutic targets for the treatment of NP (Chang et al. 2016). Epiregulin (EREG)-mediated activation of EGFR enhances nociception through a mechanism involving the PI3K/AKT/mTOR pathway and matrix metalloproteinase-9. The importance of this pathway has been demonstrated in mouse models of inflammation and chronic pain, whereas damage from NP can be significantly reduced by EGFR inhibition (Martin et al. 2017). Inhibition of EGFR activation attenuates pain sensitivity to mechanical, hot and cold stimuli in chronic compression of unilateral lumbar dorsal root ganglion (CCD)-induced rats (Wang et al. 2019). In addition, clinical studies have shown that EGFR inhibitors (e.g., cetuximab, panitumumab, gefitinib, erlotinib) improve NP (Kersten et al. 2015). Therefore, further clarification of the role of EGFR and its downstream pathways in the development of NP may help in the development of NP drugs, and EGFR may represent a potential target for treating chronic constriction injury (CCI).
Geniposide, an iridoid glycoside obtained from the dried and mature fruits of the Rubiaceae family, has been widely used in the treatment of chronic inflammatory diseases in traditional medicine Ran et al. 2021). Studies have found that it can exert analgesic function by acting on GLP-1 (Gong et al. 2014), which is known to transactivate EGFR (Gong et al. 2014) (Buteau et al. 2003;Gong et al. 2014). Geniposide also reduces the expression of inflammatory cytokines ). In addition, Xu et al. found that geniposide increases the pain threshold and relieves pain in CCI rats by inhibiting tumor necrosis factor-α (TNF-α) expression in the ipsilateral or contralateral dorsal root ganglion (DRG) (Dan et al. 2013). Although the above studies have indirectly shown that geniposide relieves chronic neuralgia, whether the inhibitory effect of geniposide alleviates NP by inhibiting EGFR has not been reported.
The effect of geniposide on NP symptoms was investigated by establishing a classical rat model of NP, i.e., CCI, and examining whether its pharmacological mechanism is related to the EGFR pathway.

Experimental Animals
Male SPF-grade Sprague-Dawley rats weighing 250-300 g were purchased from Shanghai JASJ Laboratory Animal Co. The rats were housed in a separate environment with alternating day and night periods based on a 12-12 h schedule. The room temperature was maintained at 24 ± 1 °C. The rats were fed and watered freely. Experiments were performed after 1 week of adaptation to the environment. All experimental operations were performed in accordance with the regulations of the Animal Ethics Committee of Ningbo University.

The CCI Model Construction
The rats were anesthetized using 35 mg/kg of 2% phenobarbital sodium injected into the large abdominal cavity and then fixed on the operating table. The right thigh was skinned and disinfected with iodine. The middle and back of the right thigh were dissected, and the muscle was bluntly separated. The upper middle section of the sciatic nerve was exposed, and the nerve was free to the bifurcation of the peroneal nerve. Then, 4-0 chromium catgut was wrapped around the nerve. Four mild ligature loops with a spacing of 1 mm were tied with such strength that they did not affect the blood flow of the outer membrane. The thigh muscles twitched, or the knee-jerk reflex occurred. After a local sprinkling of penicillin powder, the muscle and skin were sutured with silk disinfected sutures in sequence. The sham group underwent the same steps as the model group except that the sciatic nerve was not ligated. Five to seven days after the operation, the animals showed obvious symptoms of spontaneous pain, such as the spontaneous lifting of injured limbs and occasional licking, biting or shaking of feet, which were regarded as successful modeling. See supplement 1 for flowcharts of animal experiments.

Animal Grouping
Seven days after CCI modeling, rats with successful modeling were randomly divided into the model group, positive control group (Gab group, Jiangsu Hengrui Pharmaceutical Co. LTD), geniposide group (Gen group, cas no. 24512-63-8, purity ≥ 98%, purchased from Shanghai Macklin Reagent Company), and geniposide + EGFR agonist (NSC 228,155, cat no. HY-101084, MedChemExpress) group (NSC 228,155 group), with 10 animals in each group. The sham group and healthy control group were also established. Each group was treated as follows: normal control group: no treatment; pseudo-operated group; CCI model group: sciatic nerve ligation (see 2.2 for details); Gab group: 100 mg/kg Gab administered by gavage 6 times a week for 4 weeks on CCI model; Gen group: 10 mg/kg geniposide administered by intraperitoneal injection on CCI model; NSC 228,155 group: geniposide (10 mg/kg) + NSC 228,155 (2 mg/kg) administered intraperitoneally 6 times a week for 4 weeks.

Mechanical Sensitivity Assay
The mechanical withdrawal threshold (MWT) was measured to evaluate mechanical touch-evoked pain in rats in each group using a von Frey probe 1 day before and 7, 21, and 28 days after surgery. The experiments were performed from 8:00 AM to 12:00 PM, and the room temperature was maintained at 24 ± 1 °C. The rats were placed in the observation box for 30 min each day for the first 5 days after the formal start of the experiment. A transparent plexiglass box that was of 0.3 m in length, width, and height was placed on a 30-cmhigh shelf with wire mesh at the top. The rat was placed in the box. After the rat had adapted to the quiet, the experimenter held the von Frey probe and stimulated the rat vertically through the wire mesh lattice at the plantar midfoot of the hind limb by making it slightly s-shaped for 4-6 s. The rat showed a rapid foot lift response within the stimulation time or immediately when the von Frey probe was removed. This response was recorded as a positive response, whereas the foot lift response caused by the animal's own limb movement was not included in the positive category. The response due to the animal's own limb movement was not included in the positive category. The test was performed once every 30 s for 5 consecutive times. The average pressure value obtained from the Von Frey probe is used as the foot lift threshold. A 15 g von Frey value served as the maximum folding force, and 15 g was recorded when the measured value was greater than this value.

Measurement of Thermal Hyperalgesia
The paw thermal withdrawal latency (TWL) was evaluated using the plantar test apparatus 1 day before the operation and 7, 14, 21, and 28 days after the operation. The rats were placed in a glass hood to ensure that they could move freely. After the rats were quiet for 10 min, the right heel of the rat was placed on a hot plate (temperature set at 55℃), and the time of foot contraction, i.e., TWL, was recorded. The TWL was measured every 5 min, and the average of the three measurements was taken as the TWL. The upper limit of TWL was set at 25 s to prevent thermal scalding of the rats. The laboratory was kept quiet, and the room temperature was maintained at 24 ± 1 °C. For rats in each group, the right and left foot TWL values were compared. For rats between groups, the right foot TWL values were compared.

Network Pharmacology and Molecular Docking Analysis of PPI
The molecular structures of the chemical components of geniposide were downloaded from the PubChem database (http:// pubch em. ncbi. nlm. nih. gov/). The disease targets were obtained by searching the following databases: Drug-Bank (https:// go. drugb ank. com/), TTD (http:// db. idrbl ab. net/ ttd/), and GeneCards (https:// www. Genec ards. org/). Protein-protein interaction (PPI) data were extracted from the STRING database (https:// string-db. org/). The protein type was set to "Homo sapiens" and STRING defined a reasonable confidence interval for the PPI data score (low confidence: 0.4, medium confidence: 0.4-0.7, high confidence: 0.9). The 3D structure of the protein was downloaded from the RCSB Protein Data Bank (http:// www. rcsb. org/) to obtain the protein (EGFR, PDB ID: 5YU9) for docking. The compounds were searched and defined for rotatable bonds using AutoDockTools 1.5.6. Molecular docking studies were performed using AutoDock Vina 1.1.2. and analyzed with PyMOL.

Transcriptome Analysis
After the rats in each group were sacrificed via an injection of an overdose of sodium phenobarbital, the L4-L5 segment of spinal cord tissue was obtained for transcriptome sequencing analysis (mainly for the differential expression of genes in the geniposide group, CCI model group, and normal control group). Detailed data are available in the parsing file package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of differentially expressed proteins were performed using the R package clusterProfiler. The most significantly enriched GO terms and KEGG pathways were filtered and displayed by p value.

HE Staining
The L4-L5 spinal cord segments and sciatic nerve of each group of rats were collected, fixed with neutral formaldehyde, dehydrated, paraffin-embedded, and finally made into 5-μm-thick tissue sections. The tissue sections were dewaxed with xylene, rehydrated with gradient concentration alcohol, stained with hematoxylin-eosin, dehydrated with conventional alcohol, cleared with xylene, sealed with neutral gum, and eventually observed and photographed under a light microscope (Leica).

TUNEL Staining
TUNEL staining was performed using the TUNEL Apoptosis Detection Kit (C1091, Beyotime). According to the instructions, sections were dewaxed in xylene for 5-10 min, switched to fresh xylene, and dewaxed for an additional 5-10 min. Slices were soaked in the following solutions: anhydrous ethanol for 5 min; 90% ethanol for 2 min; 70% ethanol for 2 min; and distilled water for 2 min. Then, 20 μg/ml DNase-free Proteinase K was added dropwise and diluted 1000 times with immunostaining wash or 10 mM Tris-HCl (pH 7.4-7.8) for 15-30 min at 20-37 °C (the optimal temperature and time of action for different tissues should be explored). Sections were incubated in 3% H 2 O 2 in PBS for 20 min at room temperature to inactivate endogenous peroxidase in the sections. Subsequently, the sections were washed thrice with PBS or HBSS. Fifty microliters of biotin labeling solution was added to the samples. The samples were incubated for 60 min at 37 °C and protected from light. Then, 0.1-0.3 ml of labeling reaction termination solution was added dropwise, and the sample was incubated for 10 min. Color development working solution was added and incubated for 30 min at room temperature.

Immunohistochemistry (IHC)
All antibodies were purchased from Abcam. Spinal cord tissue sections were rehydrated in xylene and graded ethanol solution. Antigen repair was performed in citrate buffer (pH = 6) in an autoclave at 121 °C for 20 min. Then, 3% H 2 O 2 was added for endogenous enzyme inactivation. Antibodies against p-p53 (ab239211, Abcam), p-p38 (ab4822, Abcam), p-EGFR (phospho-Y1068, ab182618, Abcam), and EGFR (ab52894, Abcam) were incubated overnight at 4 °C. After removing the primary antibody by washing, the slices were incubated with goat anti-rabbit IgG-HRP secondary antibody for 1 h at room temperature followed by conventional DAB color development, hematoxylin restaining, ethanol dehydration, xylene transparency, neutral gel sealing, and light microscopic observation. Image-Pro Plus 6.0 software was used to quantify the IHC results.

Statistical Analysis
All data are presented as the mean ± standard error. Differences between multiple groups were analyzed by one-way analysis of variance followed by Duncan's multiple range test. Differences between the two groups were measured by Student's t test using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant difference.

Geniposide Alleviates Pain and Reduces Inflammatory Factors in CCI Rats by Inhibiting EGFR
Based on the analgesic and anti-inflammatory effects of geniposide, this study further investigated whether geniposide could alleviate the pain threshold of CCI rats via EGFR. By screening the pain-related targets of geniposide (Fig. 1A-B), the main target proteins of geniposide were identified as EGFR, HRAD, TLR4, CDK2, HRAS, and BCL2L1. Combined with the results of molecular docking experiments (Fig. 1C), it was revealed that geniposide could form hydrogen bonds with amino acid residues Leu-788, Lys-745, Arg841, and Asp. On the seventh day, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of each group of rats were measured. The results showed that the MWT and TWL values decreased significantly in the model group (P < 0.01), and no significant difference was noted between the sham group and the control group, indicating that the CCI model was successfully constructed (Fig. 1D-E). By observing the results of the experimental group on Day 7, it was found that the Gen and Gab groups had significantly increased MWT values after 21 and 28 days (P < 0.01). In addition, the Gen group significantly increased TWL values (P < 0.05), whereas the Gab group exhibited significantly increased TWL (P < 0.01). After the addition of the EGFR agonist (NSC 228,155), the efficacy of geniposide was significantly reversed (P < 0.05). The expression of inflammatory factors (Fig. 1F-H) in the CCI model group was significantly higher than that in the normal control group (P < 0.01), whereas the expression of inflammatory factors in CCI rats was significantly reduced in the Gen group (P < 0.01). The above results suggest that geniposide alleviates MWT and TWL values in CCI rats potentially by inhibiting the EGFR pathway and suppressing the expression of inflammatory factors.

Transcriptome Analysis
To further explore the potential mechanism of geniposide in CCI treatment, transcriptome analysis was performed. Transcriptomic analysis showed that compared with the CCI model, 4756 genes were significantly decreased and 3264 genes were significantly upregulated in the geniposide treatment group (Fig. 2A). GO analysis results showed that compared with the normal group, the differentially expressed genes upregulated in the CCI model group were mainly associated with signal transduction, the immune system, the endocrine system, and the nervous system (Fig. 2B). Compared with the CCI model group, the differentially expressed genes downregulated in the geniposide treatment group were mainly associated with signal transduction and immune system biofunctional pathways (Fig. 2C). KEGG analysis showed that the calcium signaling pathway was significantly upregulated in the CCI model group compared with the control group (Fig. 2D). In contrast, the differentially expressed genes downregulated in the geniposide-treated group were mainly closely related to the calcium signaling pathway and PI3K/AKT signaling pathway, indicating that geniposide treatment of CCI rats was mainly achieved by regulating signal transduction, the immune system, calcium channels and the PI3K/AKT signaling pathway.

Geniposide Attenuates the Neuropathological Damage Caused by CCI
As visualized by pathological sections, the model showed a disorganized nerve bundle arrangement in the longitudinal section of the sciatic nerve and a large area of necrosis in the transverse section with a large amount of inflammatory cell infiltration (Fig. 3A-B). After treatment with geniposide, the nerve bundle arrangement was significantly improved compared with that in the model group, and inflammatory cell cytokine TNF-α in DRG neurons of CCI rats was detected by ELISA. G The expression of the inflammatory cytokine IL-1β in DRG neurons of CCI rats was detected by ELISA. H The expression of the inflammatory cytokine IL-6 in DRG neurons of CCI rats was detected by ELISA. **P < 0.01, *P < 0.05, vs. the control group or the sham group. ##P < 0.01, #P < 0.05, vs. the model group infiltration was reduced. In contrast, severe nerve damage was observed again after the addition of the EGFR agonist. The HE staining results of the L4-L5 spinal cord (Fig. 3C) showed that large sciatic nerve necrosis was observed in the model with a large number of infiltrating inflammatory cell, and the nerve bundles were disordered with obvious neuronal atrophy. Compared with the model group, the arrangement of nerve bundles was significantly improved and inflammatory cell infiltration was reduced in the experimental group after geniposide treatment. The dorsal horn of the spinal cord is an essential region for pain regulation (Esposito et al. 2019). TUNEL staining was used to detect the breakage of cell nuclear DNA during the early stages of apoptosis in cells (Fig. 3D), and TUNEL quantification showed that geniposide effectively and significantly (P < 0.05) induced apoptosis of spinal cord neuronal cells in CCI rats, which was reversed after exposure to the EGFR agonist ( Fig. 3E-G, P < 0.05). The above results suggest that geniposide attenuates CCIinduced neuropathic injury associated with EGFR inhibition.

Geniposide Reduces the Expression of Nerve Pain-Related and Inflammatory Proteins in CCI Model Rats
To investigate the effect of geniposide on the expression of nerve pain-related and inflammatory proteins in CCI model rats, inflammation-related proteins were examined in nerves. IHC results showed that p-p53 and p-p38 were significantly increased in the CCI model compared with the normal control group on postoperative Day 28 (P < 0.01, Fig. 4A-C). Compared with the model group, Gab and geniposide treatment significantly reduced the expression of p-p53 and p-p38 (P < 0.05), and this treatment effect was significantly reversed after the addition of the EGFR agonist (P < 0.05). In addition, western blot results showed that geniposide significantly reduced the relative levels of p-ERK and p-P38 in the spinal cord of CCI rats (P < 0.05, Fig. 4D-E). Geniposide significantly reduced the activation of IκB, p65 and p50 (P < 0.05, Fig. 4G-J). The above experimental results indicated that geniposide reduced the expression of inflammatory and pain-related molecules in the spinal cord of CCI rats. EGFR activation was critical to hindering the efficacy of geniposide.

Geniposide Inhibits the EGFR/PI3K/AKT Pathway
Based on transcriptome analysis, we examined the proteins related to the EGFR/PI3K/AKT pathway, and the IHC results showed that geniposide and Gab significantly inhibited the activation of EGFR in CCI model rats (Fig. 5A-B, P < 0.01). This inhibitory effect was significantly reversed after the addition of EGFR agonists (P < 0.01). Western blot results further confirmed that geniposide reduced the activation of EGFR ( Fig. 5C-D, P < 0.05) and further inhibited the activation of the downstream protein PI3K/AKT ( Fig. 5E-G, P < 0.01). The above results suggest that Geniposide alleviated nerve pain by inhibiting the EGFR/PI3K/AKT pathway.

Geniposide Inhibits Ca 2+ Channel-Related Proteins in the CCI Model
Transcriptome analysis showed that, in addition to the EGFR/ PI3K/AKT pathway, the Ca 2+ signaling pathway was also an important target of geniposide in relieving NP in CCI rats. To verify how geniposide affected the Ca 2+ signaling pathway, we detected the expression of Ca ion channel-related proteins by western blotting (Fig. 6A). The results showed that geniposide significantly reduced PKC expression in CCI rat nerves (P < 0.05, Fig. 6B), whereas geniposide downregulated CaM, CaM KII α and CaM KII δ protein expression in CCI rat nerves (P < 0.01, Fig. 6C, D, E). However, the addition of EGFR inhibitors significantly reversed this effect (P < 0.05). The above results confirm the results of transcriptome analysis and further suggest that the alleviating effect of geniposide on NP in CCI rats occurs through the inhibition of Ca 2+ channelrelated protein expression through a similar mechanism as that of Gab. The above results tentatively suggest that geniposide inhibits NP-related signaling pathways (as shown in Fig. 7).

Discussion
Neuropathic pain is clinically characterized by spontaneous pain and an increased response to stimuli. Many clinical pathologies, such as cancer, trauma, and diabetes, can lead to the development of NP (Dreyling et al. 2016). Unfortunately, NP is usually resistant to standard analgesics, which are further limited by various adverse side effects (Cavalli et al. 2019;Dreyling et al. 2016). Therefore, there is a great need to better understand the molecular mechanisms of pain hyperalgesia responses to identify new therapeutic targets and new treatments for patients with NP.
Growth factors and their homologous receptors promote pain sensitization and have been identified as therapeutic targets for NP (Borges et al. 2021;Chang et al. 2016). Geniposide reduces the expression of inflammatory factors (Cai et al. 2020). In this study, a CCI rat model was established to further explore the effect of GE on neuralgia in CCI rats through EGFR and explore other potential mechanisms.
Geniposide exhibits the potential to interact with EGFR through molecular docking. Through behavioral experiments, geniposide treatment was found to increase the MWT and TWL. Pathological results showed that geniposide reduced the expression of inflammatory factors. The number of DRG neurons was significantly reduced in the CCI group compared to the control group. CCI significantly increased the expression of p-p38, and the inhibition of p38 may reduce neuronal sensitization . Similarly, the p53-caspase-3 pathway plays a key role in the neural damage of DRG neurons (Gao et al. 2018). The analgesic effects of GE have been confirmed in several previous studies, including analgesic and anti-inflammatory effects (Gong et al. 2014). This finding is consistent with the results of the present study. It was found that upregulation of EGFR neural outgrowth occurred in various pain models (Wang et al. 2019). The present study found that the therapeutic effect of geniposide was significantly reversed when further agonists of EGFR were added, suggesting that geniposide may exert anti-inflammatory and pain threshold-enhancing effects through EGFR. The results of the present study for inflammatory factors also reflected the same results. Xu et al.  found that geniposide could increase the pain threshold and reduce pain by inhibiting tumor necrosis factor-α (TNF-α) expression in ipsilateral or contralateral DRG cells. Based on the molecular biological mechanisms of nerve injury, geniposide treatment may represent an effective clinical approach for pain relief. To further investigate the mechanism by which geniposide alleviates neuralgia in CCI rats, transcriptome analysis was performed on the normal group, the CCI model group, and the geniposide treatment group, and the results showed that the CCI model mainly affected the changes in the functions related to signal transduction and the immune system. These effects were reversed by geniposide. In addition, one of the fundamental mechanisms by which signal transduction and the immune system function is through Toll-like receptors (TLRs), and Toll-like receptor signaling has also emerged as a major pathway mediating neuronal cell activation after injury and infection/inflammation. It protects and defends the host organism by initiating an inflammatory signaling cascade response in response to tissue injury, whereas TLR activation promotes damage caused by inflammation in sensory neurons, which leads to excessive pain (Lacagnina et al. 2018). Cell lines overexpressing EGFR undergo receptor-mediated apoptosis (Jackson and Ceresa 2017), and these results are consistent with those of the present study.
Based on the above studies, it is hypothesized that EGFR upregulation in microglia in the CCI rat model should promote microglial proliferation, survival and differentiation, which subsequently upregulates the inflammatory response and causes increased damage to neuronal cells via inflammatory infiltration, resulting in increased apoptosis of spinal cord cells. In addition, KEGG analysis showed that the Ca 2+ signaling pathway was significantly changed in the CCI model group compared to the normal group. Geniposide alleviates NP in CCI rats mainly through the Ca 2+ signaling pathway and the PI3K/ and EGFR. E Western blot results of the PI3K/AKT pathway. F-G Quantification of PI3K/AKT and p-AKT/AKT western blot results. **P < 0.01, *P < 0.05, vs. the control group or the sham group. ##P < 0.01, #P < 0.05, vs. the model group. Scale bar is 200 μm AKT signaling pathway. The Ca 2+ signaling pathway is a key signaling pathway for initiating and maintaining NP activitydependent central sensitization (Liu et al. 2018). Therefore, the Ca 2+ signaling pathway may represent a potential target of geniposide. Zhou et al. found that geniposide inhibits influenza through the Ca 2+ signaling pathway (Zhou et al. 2021), suggesting that geniposide targets the Ca 2+ signaling pathway to exert medicinal effects. In addition, geniposide significantly downregulated the expression of PKC, PKA, CaMK IIα, and CaMK IIδ in CCI rats, which is consistent with the results of Chen et al., who studied rhodopsin to alleviate CCI in rats . The results of the present study further confirmed that geniposide alleviates the effect on CCI through the Ca 2+ signaling pathway, which is a novel finding.
In addition to the Ca 2+ signaling pathway, transcriptome analysis showed that geniposide alleviates chronic NP through the PI3K/AKT signaling pathway. PI3K contributes to the establishment and maintenance of NP (Leinders et al. 2014). Pan et al. found that geniposide alleviates IL-1β-induced joint inflammation by inhibiting the PI3K/Akt/NF-κB signaling pathway (Pan et al. 2018), thereby representing a useful treatment for degenerative joint diseases. This finding demonstrates the potential of geniposide to inhibit PI3K/Akt. In addition, Martin et al. (Martin et al. 2017) showed that EGFR phosphorylation at Y1068 leads to PI3K-Akt signaling, a phenomenon that may be increased within the DRG of various pain models.
In conjunction with previous studies on EGFR, the present study continued to investigate the effect of geniposide on the overall EGFR/PI3K/AKT pathway. Geniposide inhibited the activation of EGFR/PI3K/AKT overall, and the pathological results and western blot results showed the same trend.
In conclusion, geniposide reduced the expression of inflammatory factors and improved the pain threshold in CCI by inhibiting Ca 2+ channel activity and EGFR/PI3K/AKT activity. B-E Quantitative results of western blot of Ca 2+ signaling pathwayrelated proteins PKC, CaM, CaM KII α and CaM KII δ. **P < 0.01, *P < 0.05, vs. the control group or the sham group. ##P < 0.01, #P < 0.05, vs. the model group

Limitations
The toxicity of geniposide was not verified in this study, so the appropriate dose and the minimum analgesic dose could not be better determined. In addition, when the EGFR pathway is assessed by western blotting, the spinal dorsal is associated with pain specifically. In future studies, the spinal dorsal horn should be assessed separately to prevent false-positive results. In addition, if conditions permit, subsequent nerve cells or glial cells can be added to verify the pathway, and the effect of geniposide can be precise to specific cells to further analyze the analgesic mechanism and lay a solid foundation for clinical application.
Author Contributions DD Z and L Y designed the study. DD Z was responsible for animal and pathology and immunohistochemical experiments. QQ C was responsible for western blotting and statistical analysis. L Y obtained the funding and drafted the original manuscript. All authors have read and approved the final manuscript.