Erianin ameliorates morphine tolerance and glioma progression through the JAK2-STAT3 pathway

Highlights

• •Erianin significantly alleviates morphine tolerance and inhibits glioma progression in mouse models, demonstrating dual therapeutic potential.
• •Mechanistic insights reveal suppression of the JAK2/STAT3 signaling pathway and downregulation of BDNF expression in dorsal root ganglia (DRG).
• •Erianin upregulates miR-375 and miR-20a, which directly target JAK2, thereby modulating neuroinflammation and opioid tolerance.
• •Multi-omics integration identifies key miRNA-mRNA interactions, providing a molecular basis for Erianin’s efficacy.
• •The study offers a novel strategy for co-managing cancer pain and tumor growth via a natural compound, supporting future translational research.

Abstract

Prolonged morphine use and glioma-induced stress have a significant impact on pain management outcomes and tumor progression. This study investigates Erianin’s potential to alleviate morphine tolerance and inhibit glioma progression through its modulation of the JAK2/STAT3 pathway. Glioma-bearing morphine-tolerant mouse models were used to evaluate Erianin’s effects on analgesia, tumor growth, and molecular pathways. Erianin administration effectively reduced morphine tolerance (50 % inhibition rate) and glioma progression (60 % inhibition rate) by inhibiting the JAK2/STAT3 signaling and suppressing BDNF expression in dorsal root ganglia (DRG). Multi-omics analysis (integrating transcriptomics and miRNA-seq data) highlighted key roles of miR-375 and miR-20a in targeting JAK2, demonstrating their critical involvement in regulating morphine tolerance and glioma-induced neuroinflammation. Further, chronic morphine use was identified as modulators of the JAK2-STAT3 pathway dysregulation. These findings uncover the potential of Erianin as a therapeutic agent. Specifically, we reveal druggable targets within inflammatory signaling cascades, providing molecular blueprints for precision interventions in pain-related oncology care.

Introduction

Morphine, renowned for its potent analgesic properties, is widely used in clinical settings for pain management, with over 50 % of patients developing tolerance within 8 days of chronic administration [1]. However, the development of morphine tolerance – particularly in patients with chronic conditions such as glioma – significantly diminishes its efficacy and necessitates higher doses. This dose escalation leads to severe side effects (e.g., respiratory depression, constipation) and an elevated risk of addiction, as observed in both preclinical models and clinical reports [2,3]. The mechanisms underlying morphine tolerance involve complex neurobiological processes, including changes in the expression of various signaling molecules and pathways within the nervous system [4]. Currently, there are no therapeutic strategies available that can concurrently address both glioma progression and morphine tolerance [5].

Brain-Derived Neurotrophic Factor (BDNF) is a critical mediator in the development of morphine tolerance [6,7]. Increased BDNF expression in the dorsal root ganglion (DRG) has been implicated in the reduction of morphine's analgesic efficacy [8]. Additionally, the JAK2/STAT3 signaling pathway plays a significant role in mediating responses to chronic morphine treatment [9,10], contributing to the development of tolerance. Understanding how these pathways are regulated could provide new insights into combating morphine tolerance. MicroRNAs (miRNAs) have emerged as important regulators of gene expression in various biological processes, including pain and opioid tolerance [11,12]. Specific miRNAs, such as miR-375 [10,13] and miR-20a [14], have been identified to target JAK2 in glioma cells, suggesting their potential involvement in the regulation of morphine tolerance through the JAK2/STAT3 pathway.

Erianin, a naturally occurring bibenzyl compound, is primarily extracted from Dendrobium chrysotoxum [15]. Recent pharmacological studies reveal its dual modulatory effects on cancer-associated pathways and nociceptive signaling networks. Beyond its established anticancer activities—including inhibition of proliferation, induction of apoptosis, and suppression of angiogenesis [[16], [17], [18], [19]]—emerging evidence suggests Erianin's capacity to attenuate chemotherapy-induced peripheral neuropathy (CIPN) by targeting TRPV1-mediated calcium influx and NLRP3 inflammasome activation [[20], [21], [22]]. This unique polypharmacology positions Erianin as a promising candidate for addressing cancer progression and its associated pain comorbidities. Research has shown that Erianin exerts its effects through multiple signaling pathways, including the inhibition of the PI3K/Akt [23] and MAPK/ERK pathways [24], which are critical in cancer cell survival and proliferation. However, its effects on pain management have never been revealed. This study investigates the effects of Erianin on morphine tolerance in glioma-bearing mice. We further elucidate the molecular pathways involved, including BDNF-mediated neuroadaptation, JAK2/STAT3 signaling dynamics, and the regulatory interplay of miR-375 and miR-20a. By exploring these mechanisms, we hope to uncover potential therapeutic targets and strategies to mitigate morphine tolerance, thereby enhancing pain management in patients with glioma and potentially other chronic pain conditions.

Materials and methods

Animal model

Male C57BL/6 mice (20–25 g) (Cat # N000295, Gempharmatech, Nanjing, China) and immunodeficiency (SCID) BALB/c-Nude mice (Cat # D000521, Gempharmatech) were used in this study, housed under standard conditions with a 12-hour light/dark cycle and free access to food and water. For evaluating the effects of Erianin on the progression of glioma, BALB/c-Nude mice were divided into four groups: the negative control (NC) group, Erianin (High 10 mg kg^-1, Medium 5 mg kg^-1, and Low 2.5mg kg^-1) groups with six mice in each group. Tumor growth was monitored bi-weekly. Mice were euthanized when tumors reached a predetermined size. The volume of the tumors was calculated using the standard formula: Volume = (Width^2 × Length) / 2. Male C57BL/6 mice were used to establish the model of Morphine tolerance in glioma mice. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC). Mice were euthanized when tumors reached a predetermined size. The volume of the tumors was calculated using the standard formula: Volume = (Width^2 × Length)/2.

Establishment of morphine tolerance in glioma mice

Glioma was induced by injecting GL261 glioma cells (1 × 10^6 cells in 5 µL PBS) into the right striatum of the mice. Morphine tolerance was induced by subcutaneous (s.c.) injections of morphine sulfate (10 mg kg^-1) twice daily for 8 days [10]. Control mice received equivalent volumes of saline. Analgesic effects were assessed using the maximum possible effect percentage (MPE %) calculated from paw withdrawal latency (PWL) tests.

Erianin treatment

Erianin was administered intrathecally (i.t.) at a dose of 5 µg/10 µL once daily along with the second, fourth, and sixth morphine injections [20,25]. Control groups received vehicle injections (Saline).

Behavioral testing

Analgesic effects were assessed using the maximum possible effect percentage (MPE %) calculated from PWL tests. The PWL was measured using a Hargreaves apparatus (Cat # 37,550, Yuyan Instruments, Shanghai, China) with an infrared radiant heat source (intensity: 30 % of maximum output). Each paw was tested three times at 5-min intervals, and the mean latency was recorded. To avoid tissue damage, a cut-off time of 20 s was applied. The order of testing was randomized. Data acquisition and analysis were performed using the manufacturer’s software. The MPE % was calculated as follows: MPE %=(Post-drug latency−Baseline latency)/(Cut-off time−Baseline latency) × 100.

Western blot analysis

DRG and spinal cord tissues were collected and homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5 % non-fat milk and incubated with primary antibodies against BDNF (Cat # 108,319, Abcam, 1:3000), JAK2 (Cat # 108,596, Abcam, 1:2000), p-STAT3 (Cat # AF4607, R&D systems, 1:1500), and GAPDH (Cat # 8245, Abcam, 1:10,000) overnight at 4 °C. After washing, membranes were incubated with HRP-conjugated secondary antibodies and visualized using ECL detection reagent (Cat # P0018FM, Beyotime, Beijing, China).

Quantitative real-time PCR (qPCR)

Total RNA was extracted from DRG tissues using TRIzol reagent and reverse-transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) on a LightCycler 480 system. Relative expression levels of miRNAs and transcripts were calculated using the 2^−ΔΔCt method, normalized to U6 snRNA and GAPDH, respectively [26,27]. The primer sequences were included in Supplementary Table S1.

RNA immunoprecipitation (RIP)

RIP was performed using the Magna RIP kit (Vazyme, Nanjing, China). DRG tissues were lysed, and the lysates were incubated with magnetic beads conjugated with anti-Ago2 antibodies. Co-precipitated RNA was extracted and analyzed by qPCR to detect JAK2 mRNA abundance.

RNA fluorescence in situ hybridization (RNA-FISH)

RNA-FISH for Localization of JAK2 mRNA and miRNAs in DRG. Dorsal root ganglion (DRG) tissues were fixed, cryoprotected, and sectioned using standard protocols. RNA fluorescence in situ hybridization (RNA-FISH) was performed using a commercial kit (Cat # Bes1001, BersinBio, Guangzhou, China) according to the manufacturer’s instructions, with modifications for dual detection. Briefly, sections were hybridized with DIG-labeled probes specific for JAK2 mRNA and the miRNA of interest, followed by sequential incubation with anti-DIG-POD antibody and tyramide signal amplification (TSA). Nuclei were counterstained with DAPI. Sections were imaged on a confocal microscope, and co-localization between JAK2 mRNA and miRNAs was analyzed using AI-driven image analysis software (Image J). Automated segmentation of fluorescence signals was combined with Pearson’s correlation coefficient quantification to statistically validate spatial relationships.

Statistical analysis

Data were expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 8. Differences between groups were analyzed by one-way ANOVA with Bonferroni correction for multiple comparisons (adjusted α = 0.05/number of comparisons) in post-hoc tests. A p-value < 0.05 was considered statistically significant. The sample size (n) = 6 for in vivo experiments and N = 3 for in vitro experiments.

Results

Erianin suppresses glioma progression in vivo

We initially investigated whether Erianin could inhibit glioma progression in vivo. The progression, volume, and weight of the tumors were monitored, revealing that Erianin decelerated tumor growth when compared to the control group (Fig. 1A – 1C, p < 0.05, p < 0.01). Moreover, immunohistochemical analysis for Ki-67 expression in the xenograft tumor models showed lower expression levels in the Erianin-treated group than in the control group (Fig. 1D and 1E, p < 0.05, p < 0.01). Importantly, Erianin had minimal effects on mice weight, suggesting low toxicity. (Fig. 1F). These findings indicate that Erianin effectively hinders glioma progression.

Fig. 1.

Erianin inhibits the progression of glioma. (A) The images derived from tumors treated with or without different concentrations of Erianin. (B) Tumor volume was examined for tumors described in (A) at different time-points. **P < 0.01, control vs all Erianin treatment group; **P < 0.01, 2.5 mg kg^-1 and 5 mg kg^-1vs 10 mg kg^-1 group. (C) Tumor weight was examined for tumors described in (A). (D and E) IHC analysis was performed to determine Ki67-positive cells in tumors depicted in (A) and quantified (E). (F) Mice weight was measured for mice described in (A). *P < 0.05, **P < 0.01 vs control group.

Erianin inhibits BDNF expression in the DRG of morphine-tolerant mice with glioma

To explore the effects of Erianin on morphine tolerance, the morphine-tolerant mice with glioma was established. From day 1, morphine initially produced strong analgesia by the repeated morphine injection. Morphine exerted an anti-nociceptive effect until day 8, which is evaluated by MPE % compared with the control groups (Fig. 2A). However, from day 4 to 8, morphine-induced MPE % declined gradually, indicating tolerance to morphine was developing (Fig. 2B, P < 0.01). The MPE % did not change in the control groups. Consistently, the expression levels of BDNF were significantly increased in a time-dependent manner in DRG, but not in spinal cord (Fig. 2C - 2E). Then we investigated the anti-nociceptive effects of Erianin on morphine-tolerant behaviors of glioma mice. Erianin was delivered i.t once daily along with the second, fourth, and sixth morphine injection. The behavioral results demonstrated that repetitive i.t. injection of Erianin remarkably impaired MPE % onset reduction from day 4 to 8 after morphine treatment compared with the control-treated groups, which did not alter the declined MPE % of morphine-injected mice (Fig. 2F). Additionally, both BDNF mRNA and protein expression were significantly reduced by Erianin treatment at the end of this model (Fig. 2G, P < 0.01). These results indicate that Erianin could ameliorate the morphine tolerance in mice with glioma.

Fig. 2.

Erianin inhibits BDNF expression in the DRG of morphine-tolerant mice with glioma. (A) The diagram of model construction and Erianin treatment. (B) MPE ( %) was measured for the model mice with different treatments as indicated. (C) BDNF mRNA level was examined in DRG of model or control mice via qPCR assays. (D) BDNF mRNA level was detected in spinal cord of model or control mice via qPCR assays. (E) BDNF protein level was determined in DRG and spinal cord of model or control mice via western blot assay. (F) MPE ( %) was measured in model mice with or without Erianin treatment. (G) BDNF mRNA level was assessed in model mice with or without Erianin treatment via qPCR assays. *P < 0.05, **P < 0.01 vs control group, ^##P < 0.01 vs Morphine+NC group.

Erianin suppresses the JAK2/STAT3 signaling pathway in DRG after chronic morphine treatment

Since the previous study has demonstrated the critical role of the JAK2/STAT3 signaling pathway in DRG after chronic morphine treatment, we wondered whether the JAK2/STAT3 signaling pathway is involved in Erianin-mediated amelioration on the morphine tolerance in mice with glioma. As shown in Fig. 3A – 3C, both JAK2 mRNA and protein expression levels were upregulated in a time-dependent manner in DRG after repeated injection of morphine, but not changed in the dorsal horn of the spinal cord. Additionally, with the treatment of Erianin, the increased JAK2 expression was attenuated in DRG after repeated injection of morphine (Fig. 3D and 3E). Consistently, the expression of p-STAT3, the downstream effector of JAK2, was also suppressed by Erianin treatment (Fig. 3E). These results demonstrate that Erianin could suppress the JAK2/STAT3 pathway in DRG of glioma mice after chronic morphine treatment.

Fig. 3.

Erianin suppresses the JAK2/STAT3 signaling pathway in DRG after chronic morphine treatment. (A and B) JAK2 expression was detected in DRG of model mice. (C) JAK2 mRNA level was measured in spinal cord of model mice. (D and E) JAK2 expression was examined in model mice with or without Erianin treatment. *P < 0.05, **P < 0.01 vs control group, ^##P < 0.01 vs Morphine+NC group.

Erianin upregulates miR-375 and miR-20a levels and thus suppresses the JAK2/STAT3 signaling pathway in the DRG of glioma mice after chronic morphine treatment

Subsequently, we explored the underlying mechanisms by which Erianin suppresses the JAK2/STAT3 pathway in DRG of glioma mice. As miR-375, miR-876–3p, miR-206, miR-185–5p, miR-920, miR-17, and miR-20a have been shown to target JAK2 [14,[28], [29], [30], [31], [32]], we wondered whether they engage the effects of Erianin on the JAK2/STAT3 pathway in the DRG of glioma mice. As shown in Fig. 4A, although all of above-mentioned miRNAs levels were reduced in the DRG of glioma mice after morphine treatment, only miR-375 and miR-20a levels were upregulated after being treated with Erianin. Then miR-375 and miR-20a levels were detected in DRG after repeated morphine treatment, 20 mg kg^-1, s.c. It was identified that although miR-375 and miR-20a were downregulated in a time-dependent manner in DRG (Fig. 4B and 4C, P < 0.01), their levels in the dorsal horn of the spinal cord were unaffected (Fig. 4D and 4E). Additionally, JAK2 expression was negatively correlated with the miR-20a and miR-375 levels (Fig. 4F and 4G, P < 0.01). Furthermore, inhibition of miR-20a or miR-375 using their antagomirs attenuated the inhibitory effects of Erianin on the JAK2/STAT3 signaling pathway in DRG of glioma mice after chronic morphine treatment (Fig. 4H). There results suggest that Erianin upregulates miR-375 and miR-20a level and thus suppresses the JAK2/STAT3 signaling pathway in DRG of glioma mice after chronic morphine treatment.

Fig. 4.

Erianin upregulates miR-375 and miR-20a level and thus suppresses the JAK2/STAT3 signaling pathway in the DRG of glioma mice after chronic morphine treatment. (A) The levels of several miRNAs as indicated were measured in model mice with or without Erianin treatment via qPCR assays. (B) MiR-375 level was measured in DRG in model mice via qPCR assays. (C) MiR-20a level was examined in DRG of model mice via qPCR assays. (D) MiR-375 level was detected in spinal cord of model mice via qPCR assays. (E) MiR-20a level was examined in spinal cord of model mice. via qPCR assays (F) The correlation between JAK2 and miR-375 expression was measured in DRG of model mice via qPCR assays. (G) The correlation between JAK2 and miR-20a expression was determined in DRG of model mice via qPCR assays. (H) The protein levels of JAK2 and p-STAT3 were assessed in DRG of model mice with Erianin treatment as well as miRNA antagomir or not via western blot analysis. **P < 0.01 vs control group, ns means no significance.

JAK2 is the direct target of miR-375 and miR-20a in the DRG of morphine-tolerant mice with glioma

Although the targeting of miR-375 and miR-20a on JAK2 has been validated in glioma cells before, it should be confirmed in the DRG of morphine-tolerant mice with glioma. Thus, JAK2 expression levels were measured in the DRG of morphine-tolerant mice with glioma which had been treated with the antagomirs of miR-375 or miR-20a. As shown in Fig. 5A (P < 0.01) and 5B, it was found that the expression levels of JAK2 were significantly upregulated in the DRG of morphine-tolerant mice treated with miR-375 or miR-20a antagomirs. Additionally, RIP analysis revealed that JAK2 mRNA abundance was significantly decreased in RNA pulled down by anti-Ago2 in DRG treated with miR-375 or miR-20a antagomirs (Fig. 5C, P < 0.01). Furthermore, RNA-FISH assays indicated that JAK2 mRNA was co-localized in the DRG of morphine-tolerant mice with glioma (Fig. 5D). These results confirm the targeting of miR-375 or miR-20a in the DRG of morphine-tolerant mice with glioma.

Fig. 5.

JAK2 is the direct target of miR-375 and miR-20a in the DRG of morphine-tolerant mice with glioma. (A) JAK2 mRNA level was measured in DRG of model mice with or without miRNA antagomir treatment. (B) JAK2 protein level was detected in DRG of model mice with or without miRNA antagomir treatment. (C) JAK2 mRNA level was examined in RNA pulled down by anti-Ago2 in DRG of model mice with or without miRNA antagomir treatment. (D) RNA-FISH analysis was performed to evaluate the colocalization of JAK2 and miRNA in DRG of model mice. **P < 0.01 vs NC group.

Upregulation of miR-20a or miR-375 level inhibits BDNF expression, elicit pain-like behavior and spinal neuronal sensitization partly by downregulating JAK2 in the DRG of morphine-tolerant mice with glioma

Then we continued to investigate the effects of miR-20a and miR-375 on the expression of BDNF. The injection of miR-375 or miR-20a antagomir could increase the expression of BDNF, which was attenuated by JAK2 siRNA treatment (Fig. 6A and 6B, P < 0.01). Additionally, as Fig. 6C and 6D (P < 0.01) results showed that injection with miR-375 or miR-20a antagomir could significantly decrease the PWL lasting for 5 days, which was rescued by co-administration of JAK2 siRNA. Notably, the hyperalgesia induced by miR-375 or miR-20a antagomir was also reversed by TrkB-Fc treatment, which is a scavenger of BDNF (Fig. 6E and 6F, P < 0.01) [33]. Therefore, our results notify that miR-375 and miR-20a could ameliorate morphine tolerance partly depending on the JAK2-STAT3 pathway.

Fig. 6.

Upregulation of miR-20a or miR-375 level inhibits BDNF expression, elicit pain-like behavior and spinal neuronal sensitization partly by downregulating JAK2 in the DRG of morphine-tolerant mice with glioma. (A) BNDF mRNA level was measured in DRG of model mice with miRNA antagomir treatment as well as si-JAK2 or not. **P < 0.01 vs NC group, ^##P < 0.01 vs miR-375 antagomir group, ^$$P < 0.01 vs miR-20a antagomir group. (B) BNDF protein level was assessed in DRG of model mice with miRNA antagomir treatment as well as si-JAK2 or not. (C) PWL was determined in model mice with miR-375 antagomir treatment as well as si-JAK2 or not. (D) PWL was determined in model mice with miR-20a antagomir treatment as well as si-JAK2 or not. (E) PWL was determined in model mice with miR-375 antagomir treatment as well as TrkB-Fc or not. (F) PWL was determined in model mice with miR-20a antagomir treatment as well as TrkB-Fc or not. **P < 0.01 vs miRNA antagomir group.

Inhibition of miR-375 or miR-20a rescues Erianin-mediated amelioration of morphine tolerance in glioma mice

We then the involvement of miR-20a and miR-375 in Erianin-mediated amelioration of morphine tolerance in glioma mice. As shown in Fig. 7A and 7B (P < 0.01), the injection of miR-375 or miR-20a antagomir could increase the expression of BDNF, which was attenuated by Erianin treatment. Additionally, as Fig. 7C and 7D (P < 0.05) results showed that Erianin treatment could significantly increase the PWL lasting for 5 days, which was rescued by miR-375 or miR-20a antagomir. Therefore, these results indicate that Erianin could ameliorate morphine tolerance through upregulation of miR-375 or miR-20a.

Fig. 7.

Inhibition of miR-375 or miR-20a rescues Erianin-mediated amelioration of morphine tolerance in glioma mice. (A) BNDF mRNA level was measured in model mice with Erianin treatment as well as miRNA antagomir or not. **P < 0.01 vs NC group, ^##P < 0.01 vs Erianin group. (B) BNDF protein level was detected in model mice with Erianin treatment as well as miRNA antagomir or not. (C) PWL was examined in model mice with Erianin treatment as well as miR-375 antagomir or not. (D) PWL was determined in model mice with Erianin treatment as well as miR-20a antagomir or not. *P < 0.01 vs Erianin group.

Discussion

In this study, we investigated the effects of Erianin on morphine tolerance in glioma-bearing mice and elucidated the underlying molecular mechanisms. Our findings demonstrate that Erianin effectively ameliorates morphine tolerance, primarily through the suppression of BDNF expression and inhibition of the JAK2/STAT3 signaling pathway in the DRG (Fig. 8).

Fig. 8.

Erianin shows significant effectiveness in alleviating morphine tolerance in glioma-bearing mice through downregulation of BDNF expression and suppression of the JAK2/STAT3 signaling pathway, mediated by the upregulation of miR-375 and miR-20a.

The development of morphine tolerance represents a major clinical challenge, particularly in patients with chronic pain conditions like glioma [34]. We observed that morphine initially provided robust analgesia, but its effectiveness declined by day 8, indicating the development of tolerance. Erianin treatment significantly mitigated this decline in analgesic effect, as evidenced by the higher MPE% in Erianin-treated mice compared to controls. This suggests that Erianin has the potential to sustain morphine's analgesic effects and delay the onset of tolerance. Recent studies have highlighted the multifaceted pharmacological effects of Erianin, including its anti-inflammatory (preclinical model), anti-angiogenic (preclinical model), and anti-tumor properties (in vitro model) [[35], [36], [37]]. These findings align with our observations and support the potential of Erianin as a therapeutic agent for modulating morphine tolerance. BDNF has been implicated in the development of opioid tolerance [38]. In our study, morphine treatment led to a significant increase in BDNF expression in the DRG, which correlated with the development of tolerance. Erianin administration resulted in a notable reduction in BDNF levels, suggesting that Erianin's ameliorative effects on morphine tolerance might be mediated through the downregulation of BDNF. This finding aligns with previous studies indicating that BDNF plays a critical role in opioid-induced neural adaptations. The JAK2/STAT3 signaling pathway has been identified as a key mediator in the cellular responses to chronic morphine exposure [9,10,39]. We found that repeated morphine injections upregulated JAK2 and p-STAT3 expression in the DRG, while Erianin treatment significantly attenuated this upregulation. This indicates that Erianin suppresses the activation of the JAK2/STAT3 pathway, thereby contributing to the reduction of morphine tolerance. Our results are consistent with other studies that have shown the involvement of JAK2/STAT3 in opioid tolerance mechanisms. Further conditional knockout of JAK2 in DRG of mice model and potential organoids can also be used to evaluate the effects of Erianin’s effects.

While BDNF is a central mediator of neuroplasticity, the interplay of JAK2/STAT3 signaling, miRNA regulation, and lncRNA-driven mechanisms further orchestrates complex cellular responses [40,41]. The JAK2/STAT3 pathway not only modulates inflammatory and survival signals but also cross-talks with miRNA networks to fine-tune gene expression. For instance, miRNAs such as miR-186 and miR-185–5p act as post-transcriptional regulators of NLRP3 inflammasome and PLOD1/Akt/mTOR pathways, respectively, as demonstrated in Parkinson’s disease and oral squamous cell carcinoma [42,43]. Concurrently, lncRNAs like KCNQ1OT1 and FOXD2-AS1 serve as molecular scaffolds or competing endogenous RNAs (ceRNAs), directly influencing pri-miRNA processing or sequestering mature miRNAs to amplify oncogenic signaling. These roles are exemplified by circ_0017552 in colon cancer, which upregulates NET1 to drive proliferation via sponging tumor-suppressive miRNAs [44]. Our study extends these paradigms to the DRG model, integrating BDNF dynamics with JAK2/STAT3 activity and lncRNA-miRNA crosstalk to unravel their collective impact on neural pathophysiology. In this study, we identified that Erianin upregulated the levels of miR-375 and miR-20a, which are known to target JAK2 [14,28,45]. The upregulation of these miRNAs was associated with the suppression of the JAK2/STAT3 pathway in the DRG. Inhibition of miR-375 or miR-20a using antagomirs reversed the effects of Erianin, further confirming their role in mediating Erianin's action. This suggests that Erianin exerts its effects, at least in part, through the modulation of specific miRNAs that regulate key signaling pathways involved in morphine tolerance. Recent studies have also highlighted the role of miRNAs in mediating the pharmacological effects of Erianin, particularly in cancer and inflammatory diseases [32,33]. Recent study has also highlighted the role of miRNAs in mediating the pharmacological effects of Erianin, particularly in cancer and inflammatory diseases [36]. AI-driven analyses of miRNA networks have further enhanced our understanding of their regulatory roles in cancer progression and drug resistance [[46], [47], [48]]. Integrating such approaches could provide deeper insights into the miRNA-mediated mechanisms of Erianin's action.

Notably, our findings on Erianin's dual modulation of pain and glioma progression resonate with emerging evidence that perioperative anesthetics influence cancer biology. Local anesthetics like lidocaine suppress tumor viability and migration through multiple mechanisms, including TRPM7 channel inhibition in breast cancer models [49], while also demonstrating broad anti-migratory effects across cancer types [50,51]. Conversely, general anesthetics such as propofol exhibit immunomodulatory properties that may reduce postoperative metastasis risk, as evidenced by suppressed inflammatory cytokines in clinical meta-analyses [52,53]. The repurposing potential of anesthetic agents—particularly their ability to target ion channels and immune pathways—represents a clinically actionable strategy [54] that could synergize with Erianin's JAK2-STAT3 suppression. In glioma patients requiring morphine analgesia, selecting anesthetic regimens with documented anti-tumor effects (e.g., lidocaine infusion combined with propofol TIVA) may amplify the therapeutic benefits observed in our study.

Our study provides preclinical evidence for Erianin’s potential to attenuate morphine tolerance by targeting BDNF and the JAK2/STAT3 pathway via miR-375 and miR-20a, suggesting a novel strategy to enhance opioid efficacy. However, clinical translation requires cautious interpretation due to unresolved safety concerns and interspecies differences. While rodent models demonstrate reduced morphine dose escalation with Erianin co-administration, potential risks—such as hepatotoxicity, off-target effects on opioid-related pathways (e.g., respiratory regulation), and species-specific variations in miRNA signaling efficacy or blood-brain barrier penetration—remain uncharacterized in humans. Future efforts should prioritize comprehensive toxicological profiling in non-rodent species, dose optimization trials to balance efficacy and safety, and validation in human neuronal or ex vivo dorsal root ganglion models to confirm conserved mechanistic targets [55]. These steps are critical to advancing Erianin toward safe, clinically relevant applications in opioid tolerance management. Beyond the JAK2-STAT3-BDNF axis, emerging evidence implicates voltage-gated sodium channels (VGSCs) as critical mediators of glioma-associated pain and tumor progression. Recent pan-cancer analyses reveal that VGSC subunits, particularly the β3 subunit (SCN3B), are dysregulated across malignancies and contribute to cancer cell invasiveness by modulating adhesion-dependent signaling pathways independent of ion conduction [56]. In glioma, β3-mediated cytoskeletal remodeling enhances cell motility and may potentiate perineural invasion—a key driver of tumor-related neuropathic pain [57]. Given that STAT3 transcriptionally regulates multiple VGSC subunits (e.g., SCN9A/Nav1.7), Erianin’s suppression of JAK2-STAT3 signaling could concomitantly attenuate VGSC-mediated hyperexcitability in nociceptive neurons and glioma invasion. Future studies should investigate whether Erianin normalizes VGSC expression in dorsal root ganglia and glioma cells, thereby bridging its antinociceptive and antitumor effects through this shared mechanism. …thereby bridging its antinociceptive and antitumor effects through this shared mechanism. Looking forward, CRISPR-based functional genomics approaches offer a powerful strategy to systematically identify novel mediators of morphine tolerance. As demonstrated in recent high-throughput screens for targeted therapy resistance [58], genome-wide CRISPR knockout or activation screening in dorsal root ganglion neurons could uncover unrecognized regulators of opioid responsiveness beyond the JAK2-STAT3-VGSC axis. Such efforts may reveal druggable targets to potentiate Erianin's efficacy or overcome compensatory resistance mechanisms in glioma-associated pain management.

Conclusion

In summary, Erianin shows promising potential in alleviating morphine tolerance in glioma-bearing mice through downregulation of BDNF expression and suppression of the JAK2/STAT3 signaling pathway, mediated by the upregulation of miR-375 and miR-20a. These results highlight the therapeutic potential of Erianin in enhancing opioid analgesia and lay the groundwork for future clinical studies aimed at improving pain management strategies for patients with glioma and other chronic pain conditions, warranting further preclinical and clinical studies.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by Guizhou Provincial Basic Research Program (Natural Science) (Qian Ke He Ji Chu-[2024] Youth 020). 2025 Hospital-Level Scientific Research Fund of Beijing Jishuitan Hospital Guizhou Hospital Fund Number: JGYYK[2025]02.

Authors' contributions

H Q and F C conceived and designed the experiments; Y G, J X, X D, and S W performed the experiments; Y G and S W analyzed the data; Y G and H Q wrote the paper; F C and J X reviewed the paper. All authors have read and agreed to the published version of the manuscript.

Ethical statements

All experimental procedures were approved by the Ethics Committee for Animal Experimentation of Beijing Jishuitan Hospital Guizhou Hospital (KT2024123101). And animal studies are reported in compliance with the ARRIVE guidelines.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

CRediT authorship contribution statement

Yi Gu: Data curation, Conceptualization. Jin Xu: Investigation, Formal analysis. Xiaoli Ding: Methodology. Su Wan: Resources. Fuying Cai: Supervision, Resources. Hai Qin: Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests, personal relationships, or professional affiliations that could influence or appear to influence the work reported in this manuscript. All authors confirm that this research was conducted in the absence of any commercial, financial, or institutional relationships that could be construed as potential conflicts of interest.