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Introduction: Spinal cord injury (SCI) often causes muscle spasticity, which can be inhibited by using calcium channel blocker. Botulinum toxin type A (BoT-A) shows therapeutic efficacy on spasticity and may exert inhibitory effects on the calcium channel. Methods: A rat model with muscle spasticity was established after SCI via contusion and compression. Different concentrations (0, 1, 3 and 6 U/kg) of BoT-A Botox were injected in the extensor digitorum longus (EDL) muscles of the right hindlimb in the muscle spasticity model. The changes of muscle spasticity and calcium level in EDL muscles were measured after the establishment of SCI-induced spasticity. Ca v 3.2 calcium channel subunit and its mutant (M1560V) were analyzed using Western blot before (input) or after immunoprecipitation with anti-FLAG antibody, and their currents were measured in motoneurons by using whole-cell voltage clamp recordings. Results: SCI induced muscle spasticity, whereas calcium level in EDL muscles and expression of Ca v 3.2 was increased in the SCI model when compared with the sham group (p < 0.05). BoT-A Botox treatment significantly reduced muscle spasticity and calcium level in EDL muscles and Ca v 3.2 expression in a dose-dependent way (p < 0.05). The ratio of biotinylated to total Ca v 3.2 was reduced in the mutant (M1560V) of Ca v 3.2 and lower than that in the wild Ca v 3.2. BoT-A Botox intervention also reduced the current values of calcium channel and the ratio in a dose-dependent way (p < 0.05). Discussion: BoT-A Botox possibly attenuates SCI-induced muscle spasticity by affecting the expression of Ca v 3.2 calcium channel subunit in the rat models. There may be multiple mechanisms for the function of BoT-A Botox. Further work is needed to be done to address these issues.

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angiogenesis. However, the underlying molecular mechanisms and predictive biomarker of Anlotinib are still unclear. Method: 437 patients with advanced NSCLC enrolled in clinical study, and 294 patients received Anlotinib therapy. Retrospectively analysis of the Anlotinibadministrated 294 NSCLC patients was performed to screen out underlying biomarker for Anlotinib-responsive patients. Transcriptome and functional assays were performed to understand the anti-tumor molecular mechanism of Anlotinib in vitro. CCL2 levels and their roles in angiogenesis were evaluated by ELISA detection, RT-qPCR quantification, and immunofluorescence assay, in vivo. Changes in serum CCL2 levels were analyzed to reveal the correlation of Anlotinib response between responders and non-responders. Result: Anlotinib therapy is more beneficial to prolong OS for NSCLC patients harboring positive driver gene mutations, especially for patients harboring EGFR T790M mutation. Moreover, our data indicated that Anlotinib-induced cell viability downregulation, cell apoptosis induction, cell invasion inhibition, cell cycle arrest, and cell migration inhibition are associated with CCL2 levels in vitro. We demonstrated that Anlotinib inhibits angiogenesis of NCI-H1975 derived xenografts model via inhibiting CCL2 in vivo. Lastly, we found that Anlotinib-induced serum CCL2 level decreases are associated with the benefits of PFS and OS, in refractory advanced NSCLC patients (n ¼ 28). Conclusion: Our study reports a novel anti-angiogenesis mechanism of Anlotinib via inhibiting CCL2 in NCI-H1975 derived xenografts model, and suggests the changes in serum CCL2 levels may be used to monitor and predict clinical outcome in Anlotinib-administered refractory advanced NSCLC patients. The biomarker of serum CCL2 alteration may guide precision therapy of Anlotinib for NSCLC patients at third-line or over third-line.

Stellate ganglion blockade (SGB) protects patients from focal cerebral ischemic injury, and transection of the cervical sympathetic trunk (TCST) in a rat model can mimic SGB in humans. The purpose of this study was to investigate the mechanisms underlying the neuroprotective effects of TCST on neuronal damage in the hippocampus in a rat model of middle cerebral artery occlusion (MCAO) in an attempt to elucidate the neuroprotective effects of SGB. The modified method of Zea Longa was used to establish the permanent MCAO model. Male Wistar rats were randomly divided into three groups: sham-operated group, MCAO group, and TCST group. The animals in TCST group were sacrificed 48 h after TCST which was performed after the establishment of the MCAO model. Proteins were extracted from the ipsilateral hippocampus and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) and peptide mass fingerprinting (PMF). The levels of N-ethylmaleimide-sensitive factor (NSF) were measured as well. The results showed that 11 types of proteins were identified by 2D-DIGE. The expressions of eight proteins were changed both in the sham-operated and TCST groups, and the expressions of the other three proteins were changed in all three groups. Moreover, the expression of NSF was higher in the TCST group than in the MCAO group but lower in the MCAO group than in sham-operated group. The ratio of NSF expression between the MCAO group and shamoperated group was -1.37 (P<0.05), whereas that between the TCST group and MCAO group was 1.35 (P<0.05). Our results imply that TCST increases the expression of NSF in the hippocampus of adult rats with focal cerebral ischemia, which may contribute to the protection of the injured brain. Our study provides a theoretical basis for the therapeutic application of SGB to patients with permanent cerebral ischemia.