Wen-Luo-Tong Decoction Attenuates Paclitaxel-Induced Peripheral Neuropathy by Regulating Linoleic Acid and Glycerophospholipid Metabolism Pathways

Abstract

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious dose-limiting toxicity of many anti-neoplastic agents, especially paclitaxel, and oxaliplatin. Up to 62% of patients receiving paclitaxel regimens turn out to develop CIPN. Unfortunately, there are so few agents proved effective for prevention or management of CIPN. The reason for the current situation is that the mechanisms of CIPN are still not explicit. Traditional Chinese Medicine (TCM) has unique advantages for dealing with complex diseases. Wen-Luo-Tong (WLT) is a TCM ointment for topical application. It has been applied for prevention and management of CIPN clinically for more than 10 years. Previous animal experiments and clinical studies had manifested the availability of WLT. However, due to the unclear mechanisms of WLT, further transformation has been restricted. To investigate the therapeutic mechanisms of WLT, a metabolomic method on the basis of UPLC- MS was developed in this study. Multivariate analysis techniques, such as principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA), were applied to observe the disturbance in the metabolic state of the paclitaxel-induced peripheral neuropathy (PIPN) rat model, as well as the recovering tendency of WLT treatment. A total of 19 significant variations associated with PIPN were identified as biomarkers. Results of pathway analysis indicated that the metabolic disturbance of pathways of linoleic acid (LA) metabolism and glycerophospholipid metabolism. WLT attenuated mechanical allodynia and rebalanced the metabolic disturbances of PIPN by primarily regulating LA and glycerophospholipid metabolism pathway. Further molecular docking analysis showed some ingredients of WLT, such as hydroxysafflor yellow A (HSYA), icariin, epimedin B and 4-dihydroxybenzoic acid (DHBA), had high affinity to plenty of proteins within these two pathways.

Keywords: TCM; UHPLC-MS/MS; Wen-Luo-Tong (WLT); metabolomics; paclitaxel; peripheral neuropathy.

Figure 1

Experimental design. Animals of Control, PTX and WLT received paclitaxel/saline injection and WLT/water pediluvium during the experiment. The PIPN model was constructed by administration of paclitaxel at 8 mg/kg by intraperitoneal injection on d1, 4, 7. WLT solution was given tropically by pediiluvium twice a day for 11 consecutive days (d0-10). The behavior assay was monitored every other day (d0, 2, 4, 6, 8, 10). The rats were finally euthanized on day 10. Plasma and spinal cord samples were collected for preparation and analysis.

Figure 2

WLT attenuated Paclitaxel-induced Peripheral Neuropathy. (A) Paw withdraw threshold (PWT) of PTX and WLT group was decreased significantly after injection of paclitaxel (8 mg/kg, i.p.). Statistical difference had been shown since day 4. Compared with PTX group, WLT attenuated the mechanical allodynia. Significant difference was shown since day 6 (p < 0.05). (B) Immunochemistry staining showed that paclitaxel induced up-regulation of CX3CL1 expression in neurons and dendrites. The expression of CX3CL1 in WLT was down-regulated.

Figure 3

PLS-DA Plots of PIPN Metabolic Disturbances. The PLS-DA plots showed clear separation between Control and PTX. (A) Positive: R²X = 0.799, Q² = 0.642. (B) Negative: R²X = 0.906, Q² = 0.847.

Figure 4

Metabolites variation analysis. (A) Heatmap of metabolite concentrations. Heatmap showed the relative concentrations of differential metabolites in each sample. (B) Metabolism Pathway Affected by Paclitaxel Treatment. The result of the significant metabolites analysis showed that paclitaxel primarily disturbed the linoleic acid and glycerophospholipid metabolisms. Linoleic acid metabolism was a preferred target pathway of paclitaxel (impact 1.0).

Figure 5

Effects of WLT on Regulating the Metabolic Disturbances. WLT rebalanced most metabolic disturbances caused by paclitaxel.

Figure 6

Interaction Networks of Enzymes and Proteins. (A) Functional interaction networks of the key enzymes were collected and input into the STRING database to analyze interactions between them (interaction score = 0.9). Pla2g15, lypla1, and plb1 were 3 most interactive enzymes. Functional interaction networks of pla2g15 (B), lypla1 (C), plb1 (D). Only proteins with highest confidence (interaction score = 0.9) were considered as possible targets network for the metabolites.

Figure 7

Biological Pathways of WLT treatment. (A) Pathway of linoleic acid metabolism. (B) Pathway of glycerophospholipid metabolism. The red stars mark potential targets of WLT in pathways.