Elucidation of the mechanism of Jinmaitong against Diabetic peripheral neuropathy based on a combined strategy of network pharmacology and molecular biology.

backgroundDiabetic peripheral neuropathy (DPN) is a common complication of type 2 diabetes mellitus (T2DM) with limited treatment options. The traditional Chinese medicine Jinmaitong (JMT) has demonstrated efficacy in treating DPN in both clinical and animal studies. It is worth noting that macrophage polarization appears to play a significant role in the onset and progression of DPN. However, whether the specific mechanism by which JMT exerts its neuroprotective effects is related to macrophage polarization still requires further in-depth investigation.

methodsT2DM model was established using Sprague-Dawley (SD) rats induced by a high-fat diet for six weeks combined with streptozotocin (STZ) injection. After modeling and drug administration, the DPN status was assessed using the von Frey test to test mechanical threshold, the hot plate test and tail flick test to evaluate thermal response latency, and the bioelectric amplifier to measure motor nerve conduction velocity. In the first batch of in-vivo experiments (Batch 1), after establishing the type 2 diabetes model, we conducted herbal formula JMT administered daily via oral gavage for another four weeks, eight weeks or twelve weeks, with each study comprising four groups: control group (CON), DPN group (DPN), low-dose JMT (7.6 mg/kg) treated group (DPN + JMT), and high-dose JMT (15.2 mg/kg) treated group (DPN + JMTH). The pharmacological effects of JMT on neurological function, neuropathology, and the levels of M1 and M2 macrophage cytokine markers were evaluated in serum and sciatic nerve, respectively. After chemical profiling of JMT by liquid chromatography coupled with high-resolution mass spectrometry, network pharmacology analysis was subsequently employed to predict the potential signaling pathways that JMT targeted in treating DPN. We further explored JMT's neuroprotective effect in a second batch of in-vivo experiments. To do this, we co-administered the JAK2/STAT3 inhibitor AG490 along with macrophage polarizing agents: LPS and interleukin-4 (IL-4). The changes of M1 and M2 macrophages in bone marrow was investigated by cytometry, while the macrophages in sciatic nerves were observed by immunofluorescence. Myelin morphology was observed with Luxel fast blue staining and transmission electron microscopy. Immunofluorescence was performed to evaluate nerve injury and regeneration, with S100 and neurofilament 160 (NF160) used to label Schwann cells and axons respectively in the sciatic nerve. The protein expressions of JAK2/STAT3 signaling in sciatic nerves were examined by Western blot.

resultsJMT significantly improved neurological function and pathological damage in type 2 DPN rats. Eight weeks after diabetes induction, DPN rats showed a significant increase in pro-inflammatory cytokines and a concurrent decrease in anti-inflammatory cytokines. JMT administration effectively restored the imbalance. Furthermore, JMT reduced the proportion of M1 macrophages while increasing that of M2 macrophages. JMT promoted the polarization of macrophages from the M1 to the M2 phenotype in both bone marrow-derived macrophages and those infiltrating the sciatic nerve, which was mediated through the suppression of abnormal activation of the JAK2/STAT3 signaling pathway.

conclusionsJMT promotes the polarization of macrophages from the M1 to M2 phenotype and alleviates neuroinflammation in T2DM rats with DPN, which is associated with inhibition of the JAK2/STAT3 signaling pathway. These findings highlight the neuroprotective potential of JMT through immunomodulatory mechanisms.