†These authors contributed equally.
The effects of Danggui Sini decoction on peripheral neuropathy in oxaliplatin-induced peripheral is established. The results indicated that Danggui Sini decoction treatment significantly reduced the current amplitude of dorsal root ganglia cells undergoing agonists stimuli compared to the model-dorsal root ganglia group (P
Peripheral neuropathy (PN) commonly occurs post the treatment of oxaliplatin (OXAL) (Kono et al., 2015; Traina, 2017). It is also caused by diabetes, toxins, or drugs (such as chemotherapeutic drugs), burns. For diabetic peripheral neuropathy (DPN) during the diabetes process, which occurs or develops in 47% to 91% diabetes patients (Sathya et al., 2017). For patients receiving neurotoxic chemotherapy, about 30% to 40% of whom would develop chemotherapy-induced peripheral neuropathy (CIPN) (Pike et al., 2012). Meanwhile, the association between burns and PN has also been proven to occur in patients with rates ranging from 2% to 52%, according to different investigations (Strong et al., 2017). Indeed, the other conditions that cause injury to the peripheral nervous system could also cause different categories of PN (Callaghan et al., 2015).
The OXAL is a third-generation anti-tumor drug that is extensively applied for treating recurrent or advanced colorectal cancer (Engstrom et al., 2009). OXAL has been proven to be the most effective chemotherapeutic strategy for advanced or metastatic colorectal cancer (Andre et al., 2004). Therefore, chemotherapy-induced PN, has an adverse-effect post the OXAL application (Kono et al., 2015; Traina, 2017). It is characterized by hyperesthesia to cold-stimulation and could decrease the life quality of patients (Argyriou et al., 2013; Checchia et al., 2017).
Yamanouchi et al. (2017) reported that systematic inflammation contributes to the occurrence of docetaxel-induced PN. Park et al. (2013) also indicated that chemotherapy-induced peripheral neurotoxicity is associated with PN in cancer patients. The clinical symptoms, such as chronic neurotoxicity and inflammations, occur in more than 60% PN patients (Ventzel et al., 2016). The OXAL-induced PN includes plenty of severe symptoms, including numbness, sensory ataxia, and even sensations of pain, all of which would be worsen following with the continued OXAL treatment in clinical (Mizuno et al., 2016). Therefore, amelioration, inhibition or prevention of the above symptoms are critical for inhibiting the subsequent side-effects of OXAL chemotherapy.
Danggui Sini decoction (DSD), an aqueous extract of Angelica sinensis, Ramulus Cinnamomi, and Radix Puerariae, has been used extensively in traditional Chinese medicine (Gao et al., 2015). The previous pharmacological reports (Qian et al., 2014; Yang, 2008) illustrated that DSD plays a role in expanding blood vessels, anti-coagulation, anti-inflammation, and analgesia. Therefore, DSD is mainly used to treat coronary heart disease, ischemic vascular disorders, venous thrombosis. Moreover, DSD could also effectively inhibit oxidative stress, apoptosis and modulate mitochondrial functions (Wolfrum et al., 2001; Yokozawa et al., 2000). We hypothesize that the DSD administration might potentiate the anti-neurotoxicity effects on peripheral neuropathy of animal models. If the hypothesis is correct, then the present study will provide an alternative anti-neurotoxic drug for treating OXAL-induced peripheral neuropathy.
A total of 30 specific pathogen-free (SPF) Wistar rats, weighting 250-250 g (male), were purchased from Tengxin BioTech. Co. Ltd. (Chongqing, P. R. China). The rats were maintained in a 12 h/12 h light/dark cycle at 23-25
A total of 24 rats were intraperitoneally injected with 4 mg/kg OXAL, according to the dosage regimen of OXAL derived in a previous study (Homles et al., 1998). The OXAL was injected into rats twice per week for 4 weeks (at day 1, day 2, day 8, day 9, day 15, day 16, day 22 and day 23, respectively). The detailed processes for establishing peripheral neuropathy rat model and sample selection were as follows: (i) intragastric administration of Danggui Sini decoction, (ii) intraperitoneal injection of oxaliplatin, (iii) chloral hydrate anesthesia, (iv) cardiac perfusion, (v) isolation of nerve and sample collection. Meanwhile, the dorsal root ganglia (DRG) was isolated according to the previous study reported (Gu et al., 2010). The whole experimental design and processes of this study were listed in Fig. 1.
The above 24 rat models were divided into 4 groups, including Model-DRG group (n = 6), rat model treated with low-dosage of DSD group (DSD-L-DRG group, n = 6), rat model treated with medium-dosage DSD group (DSD-M-DRG group, n = 6) and rat model treated with high-dosage DSD group (DSD-H-DRG group, n = 6). Meanwhile, the other normal 6 rats were employed as the control group (Blank-DRG group, n = 6).
The experimental design graph of the present study. The whole experimental processes mainly included “PN model establishment”, “DGR tissue extraction”, “evaluation of current amplitude, inflammatory DGR cells, Nissl bodies, and ultra-microstructures (using different methods)”.
Clinically, the prescribed dosage of DSD for an adult is 54 g, and low-dosage is 0.62 g/mL, medium-dosage is 1.24 dosage g/mL, high-dosage is 2.48 g/mL (Liu et al., 2017). In this study, rats in DSD-L-DRG group (with 0.62 g/mL crude-drug), DSD-M-DRG group (with 1.24 g/mL crude-drug) and DSD-H-DRG group (with 2.48 g/mL crude-drug) were intragastrically administrated with 10 mL/kg DSD according to the above dosage of adults and a previous study reported (Liu et al., 2017), with a few modifications. The DSD was administered once daily for 4 weeks. Meanwhile, the Blank-DRG group and Model-DRG group were administrated with 0.9% NaCl at a final concentration of 10 mL/kg once daily for 4 weeks.
The rats were immobilized by intraperitoneally injected with 0.7% Choral hydrate (at a final concentration of 400 mg/kg body weight) for isolating the dorsal root ganglia for following experiments. At the end of all experiments, the mice were undergone the euthanasia by intraperitoneally injecting with pentobarbital at dosage of 120 mg/kg body weight. The dorsal root ganglia were isolated by utilizing the micro-forceps and placed in D-Hank’s medium at a temperature of 4
To investigate the effects of DSD treatment on electrophysiological characteristics of the membrane, Patch-clamp electrophysiology was conducted in this study. In the present patch-clamp electrophysiology experiment, DRG cells were also treated with transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin (at a final concentration of 1
DRG tissue was isolated according to the above methods and was fixed using 4% formaldehyde (Beyotime Biotech., Shanghai, P. R. China) in PBS solution (Beyotime Biotech). Then, the DRG tissue was simultaneously embedded with the paraffin, and the histology of DRG tissues was also simultaneously visualized with hematoxylin-eosin (HE) staining based on previously published reports (Damjanov and Andrews, 2016). The HE stained DRG tissue was captured and observed using a digital microscope (Mode: DSX110, Olympus, Tokyo, Japan). Finally, the digital graphs or electronic images were collected from the representative areas with a magnification of 100
The amounts of Nissl bodies represent the status of DRG cells; therefore, the Nissl bodies were evaluated in this study. Therefore, the neuronal damages of DRG tissues were simultaneously evaluated by staining with the Nissl staining method as instruction of manufacturer (Beyotime Biotech. Shanghai, P. R. China). The paraffin-embedded DRG tissues were sliced into sections with a thickness of 0.4
DRG tissue samples (size of 1 to 1.5 mm
SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) was used for data analysis. The measurement data were presented as mean
Patch-clamp electrophysiology results showed that the current amplitude levels of low-, medium- and the high-DSD-DGR group were significantly decreased compared to the Model-GRG group (Fig. 2A, all P
Moreover, when treated with the other two agonists (menthol and mustard oil), the current amplitude levels of DGR cells were also significantly reduced compared to the Mode-DRG + menthol and Model-DRG + mustard oil group, respectively (Fig. 2B, all P
Evaluation of the current amplitude using patch-clamp electrophysiology. (A) Current amplitude for the Model-DRG and Model-DRG undergoing capsaicin stimuli. (B) Current amplitude for the Model-DRG undergoing menthol and mustard oil stimuli. Low-, medium- and high-DSD-DGR treatment significantly decreased the current amplitude compared to the Model-GRG group. The current amplitude levels of DSD-L, DSD-M, GSD-H-DRG + capsaicin group were significantly decreased compared to the capsaicin administrated groups. The menthol and mustard oil administrations also illustrated the same statistical differences. The
The inflammatory conditions could reflect the peripheral neuropathy and neurotoxicity of DRG cells; therefore, HE staining was employed to examine inflammation (Fig. 3A). The results indicated that the amounts of inflammatory cells of the Model-DRG group were significantly lower compared to the Blank-DRG group (Fig. 3B, P
Examination for the inflammatory response in DRG tissue using HE staining (n = 6 rats in each group). (A) Images of HE staining in different groups. (B) Statistical analysis for the HE staining of inflammation. The model-DRG group illustrated higher amounts of inflammatory cells compared to the Blank-DRG group, while DSD treatment decreased inflammatory cell amounts. For every group, at least 12 sections (2 sections per rat) were from rats that were used for the analysis.
The Nissl body staining results showed that, in the Model-DRG group, there were fewer Nissl bodies illustrating with damaged morphology of Nissl bodies (Fig. 4A). Meanwhile, there even more Nissl bodies were illustrating typical ellipsoidal or triangle corpuscle morphologies (Fig. 4A). The results indicated that amounts of Nissl bodies in Model-DGR group were significantly decreased compared to Blank-DRG group (Fig. 4B, P
Nissl body identification in DRG tissue using Nissl staining method (n = 6 in each group). (A) Graphs for the Nissl staining positive Nissl bodies. (B) Statistical analysis for the Nissl bodies. The black arrows represent the Nissl bodies. The model-DRG group showed lower amounts of Nissl bodies compared to the Blank-DRG group, while DSD treatment enhanced Nissl bodies of DRG cells. For every group, at least 12 sections (2 sections per rat) were from rats that were used for the analysis.
According to the electron microscopy findings, there were typical ultra-microstructures of normal neurons (such as well-developed Golgi apparatus, well-structured mitochondria) in the Blank-DRG group. However, plenty of ultra-microstructures were damaged in the Model-DRG group (Fig. 5). What’s most important is that the DSD treatment improved the ultra-microstructures of DRG cells (Fig. 5). Among all three dosages of DSD, ultra-microstructures were improved following the increased concentrations of DSD (Fig. 5). Furthermore, there were no obvious differences for ultra-microstructures among all three DSD treated groups (Fig. 5).
Evaluation of the ultra-microstructures of DRG tissue using transmission electron microscopy (n = 6 in each group). The images showed that the DSD treatment improved the ultra-microstructures of DRG cells. Magnification, 2000
We employed an effective Chinese Medicine, DSD, for treating oxaliplatin-induced side-effects in DRG cells. Clinically, the oxaliplatin-induced side-effects mainly include electrophysiological characteristic changes, inflammatory response, Nissl body depletion, and damaged ultra-microstructures, all of which are associated with peripheral neuropathy or neurotoxicity (Coriat et al., 2014; Kono et al., 2015; Naderali et al., 2018). However, all of these side-effects always occur in patients suffering from cancers and undergoing treatment of OXAL. Therefore, the novel discovered DSD might provide a promising or potential basis for OXAL-induced side-effects or complications in clinical.
Liu et al. (2017) administrated DSD at a dosage of 25-100 mg/kg for 10 days. However, in this study, the above regimen has not been illustrated the obvious effects of GDGN. Therefore, we administrated the DSD at 10 mg/kg daily for 4 weeks. In this study, the electrophysiological data of the Model-DRG group showed the abnormal current amplitudes that probably damage the physiological functions of DRG cells. The varied or aberrant currents have been illustrated in different sizes of DRG neurons (Scroggs et al., 1994). DSN treatment resulted in the enhanced excitability and reduced current amplitude of DGR cells undergoing agonists stimuli (Than et al., 2013), such as TRPV1 agonist, capsaicin (Lin and Chen, 2015), TRPM8 agonist, menthol (Tsuzuki et al., 2004) and TRPA1 agonist, mustard oil (Kistner et al., 2016). The TRPM8 is a sensory molecule expressing on the subpopulation of the primary afferent neurons and can be activated by menthol (McKemy et al., 2002). TRPV1 normally selectively expresses in small-medium DRG neurons and up-regulates in uninjured sensory neurons post the partial nerve injury (Kim et al., 2008). TRPA1, as a cold-sensor, plays roles in pain and inflammation of DRG neurons (Kistner et al., 2016). All of the above TRPM8, TRPV1 and TRPA1 molecules could modulate the intracellular Ca
Choi et al. (2006) also reported that the electrogenesis of DRG neurons is correlated with the reduced current amplitude, which could reflect the physiological functions of DRG cells. Our results indicated that the low-, medium- and high-DSD-DGR treatment, as well as DGR plus TRPM8, TRPV1 and TRPA1 agonists, significantly reduced current amplitude. These results suggest that the DSD treatment remarkably improved the physiological functions of DRG cells by reducing the current amplitudes.
Chen et al. (2014) and Yamanouchi et al. (2017) reported that inflammation is associated with the injury or damage of neurons. Therefore, HE staining was used to observe the inflammatory response in this study. Our results illustrated that DSD treatment significantly decreased the formation of inflammatory lesions in DRG tissues, with a dose-dependent efficacy. Therefore, we speculated that the effective inhibition of inflammation using DSD could result in the suppression of neuronal damages. This result suggests that the DSD remarkably alleviates the pathogenesis of peripheral neuropathy by inhibiting inflammation in DRG tissues. The functional mechanism of DSD is consistent with the report of a previous study (Yamanouchi et al., 2017).
In this study, we also observed Nissl bodies in DRG cells, which are the biomarker for the normal morphology of neurons (Niu et al., 2015). Our data showed that the DSD treatment enhanced the amounts of Nissl bodies and improved the morphology of DRG cells. Lin et al. (2017) reported that the DRG cells in painful diabetic neuropathy significantly reduced amounts of Nissl bodies. Therefore, the amounts of Nissl bodies could reflect the proliferative status of DRG cells. In this study, we confirmed that the DSD could promote DRG cell viability by improving the morphology of Nissl bodies.
Moreover, ultra-microstructures of DRG cells were also evaluated. The results showed that DSD could improve the damaged ultra-microstructures of DRG cells to be well-developed morphologies (Kerchner et al., 2012). This result suggests that DSD treatment effectively inhibits the OXAL-induced ultra-microstructure changes of DRG. The findings of ultra-microstructures in DRG cells undergoing DSD treatment are consistent with HE staining and Nissl staining results, all of which are inclined to the improvements of DRG cells.
In conclusion, DSD treatment reduced the current amplitude of DGR cells undergoing Agonists stimuli and inhibited the inflammatory response and enhanced the amounts of Nissl bodies in DRG cells. DSD treatment also improved the ultra-microstructures of DRG cells, consistent with our hypothesis. Finally, DSD did protect against the neurotoxicity of OXAL-induced peripheral neuropathy in the rat model by suppressing inflammatory lesions, improving ultra-microstructures, and enhancing amounts of Nissl bodies.
CIPN: chemotherapy-induced peripheral neuropathy; DSD: Danggui Sini decoction; DMEM: Dulbecco’s modified eagle’s medium; DRG: dorsal root ganglia; EGTA: ethylene glycol-bis (
RD and JGH designed the research study. RD, YW, JPZ, WGL, and GLW performed the research. ZCG made the statistical analysis. ZTA conducted the literature review.
The present study was conducted according to the Guidance of Care and Use of Laboratory Animals of the National Institute of Health (NIH). All of the animal experiments were approved by the Ethics Committee of Nanjing University of Chinese Medicine, Nanjing, P. R. China. All experiments involving rats were conducted based on Institutional Animal Care and Use Committee Guidelines of Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing, P. R. China (Approval No. AEWC-20180326-29).
This study was granted by the National natural science foundation of the People's Republic of China (Grant No. 81503568), the National natural science foundation of Jiangsu Province (Grant No. BK20151049) and Bureau of Traditional Chinese Medicine of Jiangsu Province of China (Grant No. YB2015035).
The authors declare no competing financial or commercial interests in this manuscript.