According to the International Diabetes Federation, approximately 537 million adults aged 20–79 years were diagnosed with diabetes by 2021. Diabetic peripheral neuropathy (DPN) is a common complication occurring in >50% of the patients with diabetes (Tracy and Dyck, 2008). DPN is often neglected but is serious and can impair sensory and motor neurons of the peripheral nerves, leading to pain and numbness in the lower limbs, and is the leading cause of lower-limb amputation (Burgess et al., 2021). Despite its prevalence, the prevention and treatment of DPN remain challenging. General clinical treatment regimens for DPN include glucose control, pain management, and neurotrophy. Glucose control was effective in decreasing the incidence of DPN in patients with type 1 diabetes but had little effect on patients with type 2 diabetes, which accounts for ∼90% of all diabetes cases (Boussageon et al., 2011; Callaghan et al., 2012). Although the pathogenesis of DPN remains unclear, Schwann cell (SC), as the most abundant cells in the peripheral nervous system, is susceptible to high glucose and lipid levels. During the occurrence and development of DPN, many key signaling pathways in SC were activated, such as oxidative stress (Pang et al., 2020; Fan et al., 2021), inflammation (Cheng et al., 2020; Du et al., 2020), endoplasmic reticulum stress (Li et al., 2017; Yang et al., 2022), and dyslipidemia (Zhu et al., 2019; Wang et al., 2020; Semler et al., 2022), and ultimately induced diabetic neuropathy (Gonçalves, et al., 2017). SC dysfunction was a major contributor to DPN, thus SC was considered as an important therapeutic target of DPN (Naruse, 2019; An et al., 2021a).

High-glucose (and high-lipid) injured SC is a recognized in vitro cell model of DPN which is usually used to evaluate the therapeutic effect, explore the mechanism of action and discovery potential medicines of DPN (Han et al., 2014; Cheng et al., 2020; Fan et al., 2021; Zhang et al., 2021b; Yang et al., 2022; Yuan et al., 2022; Pang et al., 2023). Majd et al. (2023) derived SC from human pluripotent stem cells and screened out a therapeutic candidate for DPN, bupropion, which was verified that it could prevent DPN in vivo, by performing a high-throughput screen on high-glucose induced SC injury model (Majd et al., 2023). Therefore, SC injury model is effective to screen the active constituents for treating DPN.

Traditional Chinese medicine (TCM) has unique advantages in treating complex chronic diseases. TCM has unique superiority in the treatment of DPN (Zhao et al., 2013; An et al., 2021b), and Mudan granules (MD) is a representative Chinese patent medicine. MD can alleviate limb pain and numbness, and increase nerve conduction velocity (Zhang et al., 2021a; Zhang et al., 2021c). MD is composed of nine Chinese medicinal herbs, including the radix of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. (Huangqi in Chinese), rhizome of Corydalis yanhusuo W.T. Wang (Yanhusuo), radix and rhizome of Panax notoginseng (Burk.) F. H. Chen (Sanqi), radix of Paeonia lactiflora Pall. or Paeonia veitchii Lynch (Chishao), radix and rhizome of Salvia miltiorrhiza Bge. (Danshen), rhizome of Ligusticum chuanxiong Hort. (Chuanxiong), flowers of Carthamus tinctorius L. (Honghua), lignum of Caesalpinia sappan L. (Sumu), and caulis of Spatholobus suberectus Dunn (Jixueteng). The accepted Latin names of plants were confirmed through the World Flora Online (https://www.worldfloraonline.org/) and listed in Table 1. Pharmacological experiments showed that MD alleviated peripheral nerve injury in DPN and diabetic rats (Qi, 2015; Chen et al., 2017), and improved SC morphology in the sural nerve (Qi, 2015). However, the protective pathways of MD on SC and its effective constituents have not been investigated.

Chemical constituents are the basis of effect. To the best of our knowledge, only two studies have reported the chemical constituents of MD. Tian et al. (2022) determined the nine constituents of MD using high-performance liquid chromatography (HPLC) (Tian et al., 2022). Ren (2018) determined tetrahydropalmatine content using ultra-performance liquid chromatography (UPLC) (Ren, 2018). Therefore, the constituents in MD need further investigation. Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is a powerful and widely used tool for identifying the constituents of TCM (He et al., 2018; Chen et al., 2019; Katragunta et al., 2019; Wang et al., 2019a; Liu et al., 2021); however, rapid analysis of MS data remains challenging. Molecular networking (MN), which was first proposed for MS data analysis in metabolomics (Nguyen et al., 2013; Yang et al., 2013), is based on MS2 spectra similarities determined through global natural product social molecular networking and can be used in the rapid identification of chemical constituents in natural products (Wolfender and Allard, 2016; Wang et al., 2019b; Jin et al., 2021).

This study investigated the constituents of MD using UPLC-QTOF-MS coupled with MN, and elucidated effective constituents and protective pathways of MD against high-glucose/lipid injury in SC.