目前，氫氣被證明具有腫瘤抑制的效果，但是存在治療效率低或體內(nèi)生物安全性等問題。本研究使用鎂基生物材料在腫瘤環(huán)境中的降解，設(shè)計了一種可控的局部氫氣釋放方法，具有高效的腫瘤抑制效果和良好的生物相容性，進(jìn)一步依據(jù)mRNA轉(zhuǎn)錄組測序結(jié)果揭示氫分子誘導(dǎo)腫瘤凋亡的具體作用機制。

01、研究內(nèi)容簡介

近十年來，氫分子被發(fā)現(xiàn)具有疾病治療效果，如炎癥、腦損傷、阿爾茲海默癥等。目前，氫分子醫(yī)學(xué)被廣泛接受的作用機制是降低了具有毒性的活性氧的水平，如過氧亞硝基、羥自由基等。但是，氫分子針對特定疾?。ㄈ缒[瘤）的完整作用機制通路仍不明確。

傳統(tǒng)氫氣由機體攝取的方式包括呼吸道、消化道、直接注射。這些氫氣攝取方法對體內(nèi)深部腫瘤的治療效果有限，因為氫氣具有高擴(kuò)散性、低溶解性等特點，無法在腫瘤周圍實現(xiàn)較高的溶解氫氣濃度。為了解決這些問題，局部氫氣釋放的治療策略被相繼開發(fā)，如可以釋放氫氣的納米顆粒直接注射或靶向到腫瘤組織，實現(xiàn)腫瘤周圍較高的氫氣富集，提高治療效果。然而，這些具有不可降解性和細(xì)胞毒性的納米顆粒也增加了患者健康風(fēng)險。

作為可降解金屬，鎂具有相對較低的電極電位可以與人體內(nèi)的體液反應(yīng)生成氫氧化鎂并釋放氫氣。同時，大量前期結(jié)果證明，鎂金屬植入物在動物或人體內(nèi)具有生物安全性和良好的生物相容性。生物鎂金屬材料在體內(nèi)可以創(chuàng)造一個高飽和氫氣環(huán)境，在鎂植入物周圍氫氣濃度可達(dá)到1.46 mM，高于大氣壓下水中飽和氫氣濃度0.6 mM。盡管鎂金屬作為氫氣載體在腫瘤疾病中治療效果已被報道，但是氫分子如何誘導(dǎo)腫瘤細(xì)胞凋亡和相關(guān)作用通路仍需要進(jìn)一步探究。

在本研究中，通過改變腫瘤弱酸性微環(huán)境和鎂金屬暴露在腫瘤周圍的表面積，實現(xiàn)有效調(diào)控鎂植入物的氫氣釋放速率的目的，通過使用不同濃度的氫氣處理結(jié)直腸癌細(xì)胞，探索其誘導(dǎo)腫瘤細(xì)胞凋亡的氫氣濃度范圍，并在體內(nèi)荷瘤小鼠模型中進(jìn)一步驗證。此外，本研究還通過對空白對照組和氫氣處理組的細(xì)胞進(jìn)行mRNA轉(zhuǎn)錄組測序，對比分析鎂釋放氫氣抑制結(jié)直腸腫瘤的潛在機制。

實驗結(jié)果表明，在腫瘤組織周圍酸性環(huán)境中通過調(diào)整鎂植入物表面積，可以在體液中控制氫氣的釋放速度，從而調(diào)節(jié)局部的氫氣溶解濃度，實現(xiàn)局部可控的高效氫氣輸送方式 (Fig. 1)。

Fig.1 Controlled release kinetics of hydrogen from Mg. (a) The volume of released H2 and (b) the concentration of dissolved H2 in PBS solution increased with the exposed surface area (mm2 per mL PBS) of Mg plates (PBS: pH = 7.4); (c) the volume of released H2 and (d) the concentration of dissolved H2 in PBS increased as the pH value decreases.

使用氫氣釋放裝置單獨研究了鎂的降解產(chǎn)物氫氣對人結(jié)腸癌細(xì)胞HCT116的影響。通過活細(xì)胞成像系統(tǒng)和細(xì)胞學(xué)實驗，發(fā)現(xiàn)氫氣可以阻滯HCT116細(xì)胞分裂，誘導(dǎo)凋亡，最終抑制腫瘤細(xì)胞增殖 (Fig. 2)。

Fig.2 Hydrogen induces tumor cell apoptosis at certain critical concentrations. (a) A series of morphology images of HCT116 cells from a live cell imaging microscope. (b) Schematic illustration of H2-emitting device. (c) The cell viability of HCT116 cells with different treatments. (d) The cell cycle of the HCT116 cells in the Ctrl and M-H2 groups; and (e) flow cytometry of cell apoptosis after different treatments and the corresponding apoptosis rate was statistically quantified. L-H2 and M-H2 groups represent the low and medium concentrations of H2 in the culture medium, respectively. The ctrl group represents no H2-treatment.

綜合通過MSigDB、KEGG和GO三個數(shù)據(jù)庫分析mRNA轉(zhuǎn)錄組測序結(jié)果，氫分子通過上調(diào)p53基因的表達(dá)，來調(diào)節(jié)溶酶體-線粒體誘導(dǎo)細(xì)胞凋亡 (Fig. 3)。

Fig.3 Hydrogen regulated gene expressions related to the P53-mediated apoptosis signaling pathway. (a) A volcano plot showing up/down-regulated genes after M-H2 treatment vs. the Ctrl group. Genes with an absolute fold change of >2 and a P value of <0.05 are highlighted in green and red color separately, denoting the down- and up-regulated genes, respectively. (b) Circular visualization of the gene-annotation enrichment analysis after gene set enrichment analysis (GSEA) was conducted using the Molecular Signatures Database (MSigDB). (c) The gene set enrichment analysis of the P53, lysosome and apoptosis pathway. (NES: normalized enrichment score). (d) A heat map of differentially expressed genes associated with three apoptotic pathways after H2 treatment, a fold change of >2 and a p value of <0.05 compared to the Ctrl group was analyzed. (e, f) An analysis of the enriched differentially expressed genes using the Kyoto Encyclopedia of Genes (KEGG) and Genomes (GO) database.

接下來，測序結(jié)果提示的作用通路相關(guān)蛋白表達(dá)，通過western blot和免疫熒光實驗進(jìn)行測試，來驗證氫分子調(diào)控p53誘導(dǎo)結(jié)直腸腫瘤細(xì)胞凋亡通路的有效性 (Fig. 4)。結(jié)果發(fā)現(xiàn)，氫分子提高抑癌基因p53的表達(dá)水平，促使線粒體的膜電位去極化，激活細(xì)胞自噬產(chǎn)生凋亡小體，清除了腫瘤細(xì)胞內(nèi)的活性氧，最終導(dǎo)致了腫瘤細(xì)胞的凋亡 (Fig. 5)。

Fig. 4 The up-regulated p53 activates the mitochondrial apoptosis pathway by hydrogen. (a) The p53, cathepsins, Bax, Bcl-2 and LC3B expression after M-H2, L-H2 and Ctrl treatment, respectively. (b) Confocal fluorescence images of mitochondria membrane potential and apoptosis of HCT116 with or without H2 treatment. The mitochondria membrane potential was stained by Mito-Tracker Red CMXRos (red), apoptosis cells were stained by Annexin V-FITC (green) and the nuclei were stained by Hoechst 33342 (blue). The scale bar is 25 μm. (c) The analysis of cytochrome C expression in HCT116 cells using the APC-conjugated cytochrome C antibody. Scale bar: 25 μm. (d) The analysis of the caspase-3 expression in HCT116 cells using FITC-conjugated cleaved caspase-3 antibody. Scale bar: 25 μm.

Fig. 5 Schematic illustration of the anti-tumor effect of H2 from biodegradable Mg

基于體外細(xì)胞學(xué)實驗研究結(jié)果，進(jìn)一步開展動物體內(nèi)實驗驗證氫氣的抗腫瘤效果，并比較鎂植入物腫瘤周圍原位釋放氫氣和吸入氫氣這兩種氫氣輸送方式對腫瘤的抑制率。結(jié)果顯示，鎂植入物在體內(nèi)均勻地釋放氫氣，通過控制鎂與體液的暴露面積，實現(xiàn)腫瘤周圍可控的溶解氫氣濃度影響腫瘤治療。在植入24天后，當(dāng)鎂與體液接觸面積為51.9 mm2時，鎂局部釋放氫氣比全身性吸入氫氣表現(xiàn)出更高的腫瘤抑制效果，腫瘤體積降低了27.0% (Fig. 6)。

Fig. 6 Hydrogen released from Mg wires inhibits tumor growth in vivo. (a) The anti-tumor evaluation of the H2 therapies through Mg degradation on tumor-bearing mice. (b) Micro-CT images of Mg wires after 0 d, 8 d, 16 d, and 24 d implantation in tumor tissue; (c) Evolution of hydrogen released and wire volumes in tumor-bearing mice after 8 d, 16 d and 24 d implantation. (d) Growth curve of tumor volume after different treatments. (e) The HCT116 tumor weight and the corresponding representative pictures of tumors at the end of the experiment. (f) Bodyweight change after different treatments.

通過對不同處理組的腫瘤組織進(jìn)行TEM超微組織觀察、H&E染色、TUNEL染色及caspase-3染色，鎂局部釋放氫氣表現(xiàn)出良好的體內(nèi)誘導(dǎo)腫瘤細(xì)胞凋亡效果 (Fig. 7)。

Fig. 7 Hydrogen induces tumor tissue apoptosis in tumor-bearing mice. (a) TEM photos of the tumor tissues at various scales; (b) H&E images of tumor tissues dissected from each group after different treatments on day 24; (c) Apoptosis was analyzed by TUNEL staining in tumor tissue after 24 days of treatment; (d) Confocal laser scanning microscopy of caspase-3 staining in tumor tissues after different treatments.

總之，本研究發(fā)現(xiàn)可降解鎂金屬局部釋放氫氣呈濃度依賴性誘導(dǎo)結(jié)直腸腫瘤細(xì)胞HCT116凋亡。通過控制鎂的降解速率來調(diào)節(jié)溶解在體液中的氫的濃度，例如改變介質(zhì)pH值或暴露于溶液的表面積。氫分子上調(diào)腫瘤抑制因子p53表達(dá)，通過溶酶體-線粒體介導(dǎo)的凋亡信號通路誘導(dǎo)腫瘤細(xì)胞凋亡。荷瘤小鼠體內(nèi)的鎂植入實驗也證實了其原位釋放氫氣的抗腫瘤治療效果。這些結(jié)果表明，醫(yī)用鎂植入器械（如吻合釘）控制釋放氫氣對抑制結(jié)直腸腫瘤具有很大的應(yīng)用潛力。