Bogijetong decoction and its active herbal components protect the peripheral nerve from damage caused by taxol or nerve crush

The objective of the present study was to investigate whether BGJTD was effective for protecting the peripheral nerves from damage caused by taxol injection and physical injury. Taxol generated structural disruption of the nerve, and hampered Schwann cell survival adversely. Morphological and biochemical analyses of the sciatic nerve and DRG tissue revealed that taxol and crush injury generated nerve damage in a similar way. Importantly, these pathological injuries were largely recovered by BGJTD treatment. By sorting out the herbal components of BGJTD in terms of their activity to enhance neurite outgrowth, we selected the active herbal components, and a decoction Be from selected drugs was as effective as BGJTD in facilitating neurite outgrowth and responding to heat stimulation.

Since the initial discovery and pharmacological characterization, taxol has been used as a chemotherapeutic drug. However, together with several other anticancer drugs such as platinum compounds, taxanes, vinka alkaloids, bortezomib, and thalidomide, a large portion of patients given taxol therapy showed neuropathy accompanying severe pain [21, 22]. Considering stabilization effect of taxol on microtubule assembly, axonal cytoskeletal structures mainly composed of microtubule assembly might be damaged by taxol therapy. Studies with animals and in vitro culture showed that taxol caused inhibition of neurite outgrowth of cultured neurons, disrupted myelin structure, and retarded conduction of action potential [7–9].

After peripheral nerve injury, neuropathy can occur during repair process [2, 23]. BGJTD has been used for the treatment of neuropathic pain and regulation of blood glucose levels in diabetic patients in the clinical traditional medicine, implicating the possible ameliorating effects of BGJTD on diabetic or anticancer-related neuropathy [11, 12]. To understand biological basis of BGJTD action, we performed experiment using two models of peripheral nerve injury, and reached following conclusions; first, we found that taxol injection not only inhibited neurite outgrowth of cultured DRG neurons but also induced morphological abnormalities resulting in apoptotic death of primary Schwann cells. Taxol may have a detrimental effect on axonal regrowth by inhibiting Schwann cell interaction with axons [24]. Furthermore, functional degeneration of Schwann cells by taxol injection may prevent axon myelination and lead to morphometric alterations in action potential propagation [25, 26]. Secondly, direct effects of taxol on the sciatic nerve axons were most clearly seen by immunohistochemical analysis in which ?III tubulin-labeled axons were largely degenerated by taxol injection. The structure of ?III tubulin-labeled DRG neurons at lumbar 5 was severely disintegrated by taxol injection, and phospho-Erk1/2 signals in DRG neurons were largely decreased by taxol. Previous studies have shown that phospho-Erk1/2 is induced in the peripheral nerve after injury, and can be transported retrogradely into the cell body where it leads to expression of target protein involved in axonal repair [27–29]. Here, taxol that may cause structural derangement from axon to soma could disrupt retrograde transport of Erk1/2 signaling, as has been shown by retarded transport of tracer in taxol-treated nerve [30]. Finally, BGJTD administration produced regenerative responses in the peripheral nerve. Signal intensity of apoptotic marker protein caspase 3 in taxol-treated Schwann cells was reduced by BGJTD, and Cdc2 activity, which was induced in the sciatic nerve after taxol injection or crush injury, was upregulated in the distal portion of the nerve. Moreover, neurite extension in cultured DRG neurons and axonal staining of in vivo sciatic nerves were improved in the BGJTD-treated groups, compared to corresponding taxol-treated ones.

We have reported that vimentin phosphorylated by Cdc2 kinase is induced in Schwann cells from the injured nerve and involved in regenerative responses [19, 20]. Here, Cdc2 and phospho-vimentin were detected from taxol-injected sciatic nerve, and interestingly, their levels were further upregulated by BGJTD treatment. Given that Cdc2 activation in Schwann cells is responsible for axonal regeneration, BGJTD-mediated Cdc2 induction in Schwann cells may facilitate repair process in the taxol-treated nerves. Indeed, neurite outgrowth in the taxol-treated DRG neurons was recovered by BGJTD to the level of control ones, and in vivo axon morphology, as identified by ?III-tubulin staining, was clearly improved by BGJTD. The protective effect of BGJTD was also shown in Schwann cells in which caspase 3 signals induced by taxol was largely decreased by BGJTD treatment. Thus, protective activity of BGJTD in both individual axons and Schwann cells may improve nerve repair, as demonstrated by increases in axon staining in the distal nerve stump and retrograde labeling of DRG sensory neurons (Fig. 6).

Our data showing the induction of Cdc2 and phospho-vimentin levels by BGJTD suggest possible molecular basis on how BGJTD acts on neuropathy. The BGJTD treatment upregulated Cdc2 levels and its phosphorylation of vimentin in taxol-injected nerves and the distal stump in the injured nerve. It was previously reported that Schwann cell activation in terms of Cdc2-vimentin pathway is beneficiary to promote axonal regeneration of the peripheral nerve [20]. After peripheral nerve injury, axons in the distal stump degenerate and regrow from the proximal end, and in this process, Schwann cells are actively involved in eliminating degenerating debris and guiding the growth cones at the front [24]. Molecular factors that are expressed from activated Schwann cells including growth factors and anti-inflammatory cytokines may play a role in alleviating peripheral nerve neuropathies [31, 32]. Timely interaction of Schwann cells with axons is important for recovering distal portion of injured nerve with minimizing inflammation [33]. It was reported that activation of Cdc2-vimentin pathway in Schwann cells was linked to activation of membrane intergrin, which is further involved in intercellular communication with regrowing axons [20, 24]. Whether BGJTD therapy is functionally associated with Schwann cell communication via integrin remains to be determined.

BGJTD is a decoction composed of 18 different herbal drugs and has been used in Asian medicine to treat neuropathy caused by diabetes and anticancer therapy [11]. A decoction shengmai san, which is used for the treatment of cardiovascular disorders in traditional medicine, shares the herbal component Ginseng radix with BGJTD and was reported to be involved in promoting regenerative responses after spinal cord injury [34]. Ginsenoside components such as Rg1 and Re were reported to facilitate axonal regeneration after peripheral nerve injury, or neuronal survival [35, 36], and ginsenoside Re was shown to activate Schwann cells in the injured nerve thereby promoting axonal regeneration [37]. However, given that BGJTD contains diverse herbal components, it is difficult to determine whether the efficacy of BGJTD is related to combinatorial activity or due to some specific components. Here, as an initial step to screen the possible active herbal components, we categorized 18 individual drugs into 4 groups based on description of the traditional medicine, and selected the most active components inducing neurite outgrowth of cultured neurons; a similar procedure was applied previously to search herbal drugs for therapeutic application to neurodegenerative diseases [38]. Our data showed that Be decoction composed of 4 active herbal drugs is as effective as BGJTD in inducing heat sensitivity of the hind paws. Further studies are critical to determine whether Be extract is comparable to BGJTD in regulating regenerative responses in association with peripheral nerve neuropathy.