Aqueous extract of Chuan Xiong Rhizoma enhances inhibitory effect of temozolomide against brain metastasis of melanoma in mice

doi:10.12122/j.issn.1673-4254.2024.06.09

Abstract

Abstract:

Objective To investigate the effect of the aqueous extract of Chuan Xiong Rhizoma (CR) on brain metastasis of melanoma B16F10 cells in mice. Methods C57BL/6J mouse models of brain metastasis of melanoma were established by ultrasound-guided intraventricular injection of Luc-labeled B16F10 cells, and brain tumor growth was monitored by in vivo imaging. The mouse models were then randomized for daily gavage of saline or aqueous extract of CR (equivalent crude drug concentration of 1 mg/g). Behavioral tests were used to evaluate the neuroprotective effects of CR in the tumor-bearing mice, and the changes in proteins associated with blood-brain barrier integrity, neuronal cell proliferation and apoptosis, and microglial cell apoptosis and activation were observed using immunofluorescence assay. The efficacy of CR combined with temozolomide (25 mg/kg) against brain metastases of B16F10 cells was observed by in vivo imaging. Results CR-treated mouse models did not show obvious progression of brain metastases and had a reduced rate of body weight loss and lowered protein expressions of ZO-1, claudin-5, occludin, P-gp, TNF‑α, AQP4 and PDGFRβ. In the behavioral tests, the CR-treated mice showed prolonged stay on the wooden stick with a shortened time of sticky stick removal. Immunofluorescence assay showed increased proliferation and decreased apoptosis of neuronal cells and microglia in CR-treated mice. CR treatment significantly increased the levels of CD86, CD206, IL-4 and IL-10 and decreased the levels of CD163 and IL-1β in the microenvironment of brain metastases. The mice receiving combined treatments with CR and temozolomide showed significantly lower intensity of fluorescent signals in the brain than those treated with temozolomide alone. Conclusion CR does not promote brain metastasis of melanoma while inducing opening of the blood-brain barrier, and its combined use with TMZ results in enhanced inhibition against brain metastasis of melanoma B16F10 cells in mice.

Key words: Chuan Xiong Rhizoma, Melanoma, temozolomide, blood-brain barrier, brain metastases

Qian ZHAO, Zhen ZHANG, Xingqi ZHOU, Xiangyu RONG, Xuerou LIU, Xinli ZHAO, Haojie WANG, Jinlong PANG, Shanshan LI, Xian LI. Aqueous extract of Chuan Xiong Rhizoma enhances inhibitory effect of temozolomide against brain metastasis of melanoma in mice[J]. Journal of Southern Medical University, 2024, 44(6): 1088-1097.

Figures/Tables 4

Fig.1 The aqueous extract of Chuan Xiong Rhizom (CR) does not affect survival, body weight or intracranial tumor growth in C57BL/6J mice. A: Bioluminescence imaging scan 6 days after intracardiac injection of the tumor cells (n=6). B, C: Bioluminescence imaging of intracranial tumors in C57BL/6J mice on day 6 and day 12, respectively (n=8). D: Quantitative analysis of intracranial tumor signals of bioluminescence imaging BLI (n=8). E: Quantification of intracranial tumor signals in control and CR-treated mice on day 12 (n=8). F: Kaplan-Meier survival curves of control and CR-treated mice (n=8). G: Body weight changes of control and CR-treated mice (n=8).

Fig.3 Neuroprotective effect of CR in mice with brain metastasis of B16-F10-Luc cells (×200). A: Vertical pole test for assessing forelimb grasping ability (n=10). B: Sticky dot test for assessing sensory abilities (n=10). C: Immunofluorescence staining of neuronal cells (red) and quantification of mean fluorescence intensity. D: Immunofluorescence staining of Tunnel (green) and quantification of mean fluorescence intensity. E: Immunofluorescence staining of TMEM119 (red) and cleaved caspase3 (green) and quantification of mean fluorescence intensity. *P<0.05, **P<0.01 vs control or Normal.

Fig.4 CR regulates the expression of CD68, CD86, CD163, CD206, IL-1β, IL-4, IL-6 and IL-10 (A-H,reepectively) in the brain of the tumor-bearing C57BL/6J mice （×200）. n=6, *P<0.05, **P<0.01 vs Control.

Fig.5 CR enhances the inhibitory of temozolomide against brain metastasis of melanoma B16F10 cells in mice. A: Bioluminescence imaging of intracranial tumors in C57BL/6J mice on day 13. B: Quantitative detection of intracranial tumor signals. C: Kaplan-Meier survival curves in both groups. D: Body weight changes in both groups. E: Vertical pole test for assessing forelimb grasping ability of the mice. F: Sticky dot test for assessing sensory abilities of the mice. n=17, *P<0.05, **P<0.01 vs Control.

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