Matrine induced vacuoles of human leukemia cells, and the formation of vacuoles is closely related to inhibition of cell proliferation. There is increasingly evidence that cytoplasmic vacuolization of human tumor cells by small molecules has a propulsive effect on inducing their death [30, 31]. Rac1 activated Arf6-GAP and GIT-1 proteins, to impair the function of Arf6-GTP protein, and leads to inactivation of Arf6 protein to induce vacuolation . Doxycycline induced vacuolation in cancer cell lines DU145, MDA-MB-468 and A549 cells by activated H-Ras and increased Rac1 expression, the downregulation of Rac1 reversed cytoplasmic vacuolation and cell death . In glioblastoma cells, Mitochondrial membrane potential and ATP levels increased by up-regulated H-Ras. These changes disrupt cellular metabolic function and prevent ATP-mediated fusion of late endosomes and lysosomes, and eventually the continuous accumulation of vacuoles destroys cell integrity and ultimately leads to cell death . Similarly, matrine also inhibited cell proliferation by inducing vacuolation in leukemia cells.
By overexpressing the lysosomal labeled molecule LAMP1 fused with eGFP, a large amount of green fluorescence was observed at the cell vacuolar edge after matrine treatment by fluorescence microscope, The result confirmed that the vacuoles originated from lysosomes, but not mitochondria or Golgi bodies. Recent evidence (Zhang et al., 2010)  believed that matrine-induced were autophagic vacuoles in liver cancer cells. In this study, autophagosomes were considered to be spherical structures with double or multilayer membranes, containing cytoplasmic components with a diameter of 100-900nm. Matrine-induced autophagosomes were shown in Fig. 3C, which did inconsistent with the study. The researches (Wang et al., 2013)  have also confirmed that matrine enter the lysosome of tumor cells and increase the pH of the lysosome.
In addition, the result found that the 5.3 ± 0.7 nm gold NPs18, 29, 32 functionalized with positively charged N, N, N-trimethyl (11-mercaptoundecyl) ammonium chloride (TMA) and negatively charged 11-mer-captoundecanoic acid (MUA) ligands. After co-culture with tumor cells, and targeting the lysosome, nanoparticles aggregate in the lysosome to increase osmotic pressure, which makes the lysosome absorb water and expand, and finally the lysosome ruptures and leads to cell death, The nanoparticles TMA: MUA (4:1) of killing tumor cells, there is only cationic ligands of nanoparticles has cytotoxicity both cancer and healthy cells, only with anionic ligand nanoparticles because of not well attached on the negatively charged membrane,due to it is difficult to internalize and lost its effect on tumor destruction . This result is similar to our study, matrine may be detained after entering the lysosome, inducing osmotic changes and lysosome swelling to forming vacuoles, and the production of vacuoles also inhibits lysosome function. With the further research, lysosomes have become an important target for anti-tumor therapy, and targeting lysosomes kill tumor cells is necessary [38, 39]. Combined with the current research results, we believe that the occurrence of vacuoles leads to down-regulation lysosome proteolytic and autophagy related protein accumulation, which leads to lysosome dysfunction and affects the proliferation activity of human leukemia cells.
In the process of vacuolization, V-ATPase as a hydrogen donor in the acidic environment of lysosomes, and its role is to pump hydrogen ions from the lysosomal membrane into the lysosome, it keeps a high hydrogen ion concentration in the lysosome, and maintains a significant difference in the hydrogen ion concentration inside and outside the lysosome, thereby maintaining the acidic environment in the lysosome. Our study found that among the structural analogs of matrine- sophoridine had a significant effect of inducing vacuolation, while oxymatrine did not this action. After analyzing the molecular structure of matrine, it was found that matrine and sophoridine contain free amine groups that can bind hydrogen ions, while oxymatrine does not have this structure. In addition, matrine-induced cell vacuolization can be affected by changing the acid-base environment of matrine. These evidences suggest that the occurrence of cellular vacuolation is related to the protonation of matrine. In the early years, Christian de Duve also proposed the mechanism of the accumulation of lipophilic amine small molecules in the acidic environment of lysosomes: amine-containing weak base drugs and uncharged drugs enter cells by simple diffusion, they are not only transported into the cytoplasm, but also are also transported into organelles. When these molecules diffuse into acidic organelles and protonate with hydrogen ions, they are charged and trapped in acidic organelles. the higher the osmotic pressure in the organelle, and the more organelle swells, vacuoles were observed by optical microscope. With the prolongation of drug time and the increase of concentration, the vacuoles gradually become larger and eventually rupture and died [40, 41]. Therefore, we believe that the protonation of matrine may be an important mechanism for its induction of vacuoles, the inhibition of lysosomal function by matrine-induced vacuolization is one of the ways that matrine exerts its anti-leukemia effect.
As mentioned above, matrine enters the lysosome, On the one hand, the acidic environment of the lysosome makes matrine protonated in the lysosome, resulting in an increase in the osmotic pressure in the lysosome, and local vacuoles absorb water and swelling to lead to cytoplasmic vacuolization. On the other hand, matrine is protonated in the lysosome to consume hydrogen ions, and the lysosome activates V-ATPase in order to maintain its acidic environment. In this study, it was found for the first time that matrine induced vacuolation of leukemia cells, and vacuolation originated from lysosome and inhibited lysosome function, which provided important reference value for matrine targeting lysosome therapy of leukemia cells with strong innovation. Based on current research results, in the future study, we should focus on matrine cause key molecular mechanism of cytoplasm vacuoles, in order to further elucidate the pharmacological effects of matrine, variety of matrine development and provide targeted lysosomes to treat cancer cell with meaningful reference, and increase the research of Chinese medicine to promote the development of traditional Chinese medicine.