Abstract: BACKGROUND: Studies have shown that paeoniflorin can improve autophagy defects in inflammatory and neurodegenerative diseases by reducing interleukin-6 levels and promoting Beclin1 expression. However, the effect and mechanism by which paeoniflorin improves bortezomib-induced peripheral neuropathy remain unclear.
OBJECTIVE: To explore the effect and molecular mechanism of paeoniflorin on inflammatory factors and abnormal autophagy to improve bortezomib-induced peripheral neuropathy.
METHODS: (1) Network pharmacology analysis: The two-dimensional structure of paeoniflorin was retrieved from the PubChem database and uploaded to the Swiss Target Prediction database for target prediction. Disease-associated targets of bortezomib-induced peripheral neuropathy were identified using the GeneCards and OMIM databases. The intersection between paeoniflorin’s predicted targets and genes related to bortezomib-induced peripheral neuropathy was defined as potential therapeutic targets for paeoniflorin intervention. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on these intersection targets. A “target-pathway” network was constructed, and key paeoniflorin targets were selected based on node degree values from both protein-protein interaction and “target-pathway” networks. Molecular docking was then conducted to validate the binding interactions between paeoniflorin and its potential targets. (2) Cell experiments: Rat pheochromocytoma cells PC12 (or human myeloma cell line MM.1S) in logarithmic growth phase were cultured in three groups: the control group was not treated with any drugs, the model group was treated with bortezomib, and the paeoniflorin group was treated with both paeoniflorin and bortezomib. After 48 hours of culture, the cell counting kit-8 was used to detect the activity of PC12 cells (or MM.1S cells), ELISA was used to detect the level of interleukin-6 in PC12 cells, immunofluorescence was used to detect the expression of LC3B in PC12 cells, western blot was used to detect the expression of LC3 I, LC3 II, and Beclin1 proteins in PC12 cells, and transmission electron microscopy was used to observe the level of autophagy in PC12 cells. (3) Animal experiment: 45 C57BL/6J mice were randomly divided into three groups: the control group (n=15) did not receive any intervention, the model group (n=15) established a bortezomib-induced peripheral neuropathy model by intraperitoneal injection of bortezomib, and the paeoniflorin group (n=15) was given paeoniflorin orally once a day for 25 consecutive days during the bortezomib-induced peripheral neuropathy model process. After administration, the thermal pain sensitivity, serum interleukin-6 levels, sciatic nerve tissue morphology, and LC3 I, LC3 II, Beclin1 expression in mice were detected.
RESULTS AND CONCLUSION: (1) Network pharmacology analysis identified 16 potential therapeutic targets of paeoniflorin for bortezomib-induced peripheral neuropathy. Network topology analysis revealed interleukin-6  and Beclin-1 as key targets, with paeoniflorin demonstrating strong binding affinity to both proteins. (2) Results from the cell experiment demonstrated that bortezomib significantly reduced viability of PC12 cells while increasing interleukin-6 levels and decreasing autophagy activity. Paeoniflorin treatment effectively reduced interleukin-6 secretion, enhanced autophagy, and improved PC12 cell viability without compromising the anti-myeloma efficacy of bortezomib. (3) Animal experiments showed that bortezomib administration significantly increased thermal pain sensitivity, elevated serum interleukin-6 levels, aggravated sciatic nerve damage, and reduced autophagy levels within the sciatic nerve , whereas paeoniflorin treatment significantly improved thermal pain sensitivity, alleviated sciatic nerve injury, reduced serum interleukin-6 expression, and ameliorated bortezomib-induced autophagy defects in mice. (4) To conclude, paeoniflorin mitigates bortezomib-induced peripheral neuropathy by suppressing interleukin-6 and restoring neuronal autophagy, without compromising bortezomib’s anti-myeloma efficacy.

Key words: paeoniflorin, bortezomib, peripheral neuropathy, autophagy, interleukin-6, multiple myeloma, PC12 cells