A Mechanistic Study Of Ginsenoside Re Promoting Peripheral Nerve Regeneration Following Sciatic Nerve Crush Injury

Objective1、To explore the clinical merit of ginsenoside Re in promoting nerve regeneration, given the fact that researches in the field have shown that ginsenoside Re exhibits multiple pharmacological activities via different mechanisms both in vivo and in vitro.2、To evaluate the effects of ginsenoside Re on the proliferation, differentiation and migration in SNC rat model.3、To uncover the relevant signaling pathways involved in the process of peripheral nerve regeneration in the SNC models following the administration of ginsenoside Re.Methods1、Adult male Sprague-Dawley rats (weighing 250-300 g) were used in our study. The rats were randomly divided into nine groups consisting of one normal group (n= 4), one saline group (n= 4), and seven sciatic nerve crush groups (n= 28). Before injured, the sciatic nerve crush groups were intraperitoneally injected different doses of Re (The concentration of Re:0,0.5,1.0, 1.5,2.0,2.5, and 3.0 mg/kg). The saline group was injected with the same volume of saline. This process lasts for 3 weeks. In the second week after intraperitoneally injection, the sciatic nerve crush groups were anesthetized with pentobarbital (50 mg/kg, i.p.). Using aseptic technique, the right sciatic nerve was exposed 1.0 cm distal to the sciatic notch by blunt dissection, then crushed by a small hemostat of 3-mmwidth at the midpoint for 10 s, and then unclamped for 10 s, repeated three times as described in previous report. The rats were allowed to recover from the surgery and housed under a 12 h light-dark cycle in the room temperature (RT). At 1 week after surgery, the animals were anesthetized to harvest the sci-atic nerves on ice, extracting nerves from the normal, saline group, and the sciatic nerve crush injury groups. At each time point, we cut 1-cm-long sciatic nerve segments cen-tered on the lesion site, and the sciatic nerves were snap frozen at -80 LC until use. Additional experimental ani-mals (normal, saline, sciatic nerve crush group-0.5, 1.0,1.5,2.0,2.5,3.0 mg/kg Re, n= 3 per group) for sections were anesthetized and perfused through the ascending aorta with 0.9% saline, followed by 4% paraformaldehyde at each time point. All surgical interventions and postop-erative animal care were executed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Chinese National Committee to the Use of Ex-perimental Animals for Medical Purposes, Jiangsu Branch. All efforts were made to minimize the number of animals used and their suffering.2、A series of motor activity assessments, including sciatic functional index (SFI) and the toe spread index (TSI) were employed to determine the physiological alterations of Rats.3、Western blot and Immunofluorescent Staining was performed to detect the index changes of peripheral nerve regeneration.4、Western blot was employed to detect the effect of ginsenoside Re on the expression of phosphorylated ERK and JNK.5、Trans-well and wound-healing assays were conducted to investigate the influence of ginsenoside Re on the migration of Schwann cells following the treatment with ginsenoside Re.Results1、Western blot revels that ginsenoside Re promoted the expression of PCNA in a dose-dependent manner following SNC.2、Histological sections were qualitatively analyzed at the proximal portion of the sciatic nerve. Re groups exhibited a similar distribution of small- and large-diameter myelinated and unmyelinated nerve fibers and Schwann cells, each surrounded by a well-defined endoneurium, and regular proportions between myelin sheath thickness and fiber diameter.3、GAP43 was obviously increased in the nerves after treated with 2.0mg/kg ginsenoside Re for 7 days compared with Normal and Saline group. In addition, using transverse cryosections, two other specific markers, S-100 (SCs marker), NF-200 (neuronal marker) were detected. S-100 exhited similar changes with that of GAP43, while the morphological change of NF-200 occurred, and became irregular and blur. Ginsenoside Re could ameliorate the expression alterations of the proteins.4、Immunofluorescent staining showed the co-localisation of Schwann cells and PCNA were notably increased after SNC.5、In SNC Model, the expression of Oct6 was markedly increased in SC cells. Also, ginsenoside Re was associated with SCs differentiation in SNC.6、The phosphorylation of ERK and JNK but not p38 was increased in SNC models when treatment with ginsenoside Re.7、Using trans-well and wound-healing assays, we observed an increased migration of, Schwann cells after the treatment with ginsenoside Re.Conclusions1、In sciatic nerve injury model, ginsenoside Re can significantly promote the proliferation, differentiation and migration of Schwann cells. Thus promotes peripheral nerve regeneration.2、In sciatic nerve crush injury model, the mechanism of ginsenoside Re promote peripheral nerve regeneration is the pathway of ERK-and JNK-dependent.