Zhenzhu Xiaoji Decoction Induces Autophagy and Apoptosis Cell Death in Liver Cancer Cells through AKT/mTOR and JAK2/STAT3 Signaling Pathway

Background Liver cancer is one of the most common digestive tumors. The prescription Zhenzhu Xiaoji decoction (ZZXJD) has a certain effect on the growth and survival of primary liver cancer. Object: This article aimed to explore the effect and molecular mechanism of ZZXJD on liver cancer SMMC-7721 cells. Method The research groups were divided into the model group, ZZXJD group, and cisplatin group. SMMC-7721 cells were treated with different concentrations of ZZXJD-medicated serum for 24 h and 48 h. The cell viability was measured with CCK8 assay, and cell morphology was observed by fluorescence microscope and transmission electron microscope (TEM). Western blot, RT-PCR, and gene chip were used to determine the protein expression level and gene expression level of cells and tumor tissues. Results ZZXJD inhibited the proliferation activity of SMMC-7721 cells in a concentration- and time-dependent manner. The morphological changes of the cell showed apoptosis and autophagy. The gene expression of protein kinase B (AKT), mammalian target of rapamycin (mTOR), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3(STAT3) were downregulated compared with the model group(p < 0.05). The nude mice experiments confirmed that ZZXJD inhibited the growth of tumors in tumor-bearing mice, and the effect increased with the increase of concentration. Conclusion ZZXJD induced autophagy and apoptosis of liver cancer cells via inhibiting AKT/mTOR signaling pathway and JAK2/STAT3 signaling pathway, thereby affecting the growth and survival of liver cancer cells.


Introduction
Liver cancer is currently one of the most common malignant tumors. e incidence rate of liver cancer is increasing year by year. According to the survey report in 2018, the incidence rate accounted for 4.7% of malignant tumors, and the mortality rate is 8.2% of malignant tumors [1]. In 2019, a research report pointed out that the mortality rate had accounted for 9.1% [2]. At present, the treatment of liver cancer focus on noncoding RNA and immunotherapy, while the long-term efficacy of monotherapy is not ideal, and the interaction between the mechanisms of combination therapy is unclear [3,4]. Lots of research showed that traditional Chinese medicine (TCM) had inhibitory effects on tumor growth, proliferation, migration, and metastasis. Integrated TCM and other treatments can reduce adverse reactions and prolong the survival of patients. It can be taken throughout the course of treatment [5]. e focus of current research is to find effective TCM and to fully define its mechanism of action. e JAK/STAT signaling pathway can be abnormally activated by cytokines and other biological processes, such as cell proliferation, differentiation, autophagy, apoptosis, and immune regulation. It is an important signaling pathway for the occurrence and development of tumors and other diseases [6]. e mTOR signaling pathway can participate in the occurrence and development of tumors by inhibiting tumor cell apoptosis or cell autophagy [7,8]. e two signaling pathways are both hot spots in tumor research. e prescription ZZXJD is composed of Ligustrum lucidum (Ligustrum lucidum Ait., fruit, nvzhenzi), Curcuma zedoaria (Curcuma phaeocaulis Val, root, ezhu), Hedyotis diffusa (Spreading Hedyotis Herba, baihuasheshecao), Prunella vulgaris(Prunella vulgaris L., cluster, xiakucao), and Licorice(Glycyrrhiza uralensis Fisch., root, gancao) [9][10][11][12]. Our preliminary research found that it had a certain inhibitory effect on growth and development of H22 liver cancer cell. Lots of autophagosomes were observed by TEM in ZZXJD group [13]. is experiment explored the effect of ZZXJD on human liver cancer SMMC-7721 cells and its molecular mechanism in vivo and in vitro, which provided new ideas and experimental basis for the research of new anticancer drugs and clinical medications.

Materials and Animals.
Human liver cancer SMMC-7721 cells were purchased from Fuheng, Shanghai, China. SD rats were identified and provided by Liaoning Changsheng Biotechnology company (Qualified number: SCXK-Liao-2020-0001). Sixty six-to-eight-week-old SD rats (240 ± 20 g) were half male and half female. BALB/c nude mice, provided by Beijing Weitong Lihua Laboratory Animal Technology (qualified number: SCKK-Jing-2016-0006), which were six-to-eight weeks old (15 ∼ 20) ±2 g weight and a total of 60 males and females. e experiment and breeding were carried out in a barrier environment, and the water and feed are sterilized. e animal study was reviewed and approved by Heilongjiang University of Chinese Medicine Institutional Animal Care and Use Committee (no. 2019051301; 2019112201). ZZXJD was provided from the First Affiliated Hospital of Heilongjiang University of Chinese Medicine (Table 1). e herbs were authenticated by a pharmacognosist from the Heilongjiang University of Chinese Medicine, in accordance with standard protocols of the Chinese Pharmacopoeia (Version 2020). e ingredients of the Chinese medicine have been tested and identified. e autophagy inhibitor was 3MA (Abmole, M2296), and the positive control drug was cisplatin (Abmole, M2223).

Cell Culture.
e DMEM complete culture medium (Boster, #PYG0103) is added with 10% FBS and 1% penicillin/streptomycin. SMMC-7721 cells were observed by inverted microscope. When the cells placed under the culture flask are about 70 ∼ 80%, the original culture medium was discarded, and the culture flask was gently rinsed with PBS. en, trypsinization was added in the flask. When the intercellular space of cells enlarged, fresh culture medium was put in the flask to finish digestion of trypsinization. After pipetting and centrifugation, the culture medium was extracted carefully on the cell upper layer, and then, the cells were made a single cell suspension by the fresh culture medium. e cells were cultured at 37°C with 5% CO 2 .

Preparation of Medicated
Serum. SD rats were bred adaptively in a sterile environment for three days, and the changes in physiological conditions such as body weight, food intake, and hair of the rats were observed. e dose of ZZXJD was 65 g·kg −1 body weight, which was calculated according to the equivalent dosage table of human and rat body surface area. e rats were administered by gavage for 3 days, and blood was taken from the abdominal aorta to prepare medicated serum.

CCK8 Method.
e mixed single cell suspension was diluted with complete culture medium to 5 × 10 4 cells/mL and inoculated in a 96-well plate at 100 μL/well. e outer circle of the well plate was PBS solution, and the well plate was moved to 37°C, 5% CO 2 for 24 h. e supernatant was discarded, and the plate was washed twice with PBS. en, 100 μL culture medium with ZZXJD-containing serum (0, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, and 20%) was added to the plate, and each group set up six re-wells. After adding the medicine, cells were incubated for 24 h or 48 h, and 10 μL CCK8 was added to each well solution, which was shaken and mixed and incubated for 2 h. OD value of each well was detected at 450 nm on a microplate reader. e proliferation activity of SMMC-7721 cells was calculated and analyzed. Cell viability was calculated by (experimental group OD value/control group OD value) × 100%.

Hoechst 33342
Staining. Cells were seeded at 1 × 10 4 cells/well in 48-well plates for 24 h before treatment. e wells were added 0.25 mL culture medium with drugcontaining serum of 0%, 5%, 10%, 15%, and 15% + 0.5 mM 3MA. e other two groups were 0.5 mM 3MA and 4 μg/mL cisplatin. After adding the different medicine, the orifice plate was placed in the incubator for 24 h. After washing with PBS twice, Hoechst33342 staining solution 250 µL was added to each well and placed in an incubator for 30 min. e morphology changes of SMMC-7721 cells were observed under fluorescent inverted microscope.

e Ultrastructure of Cell.
Based on Hoechst 33342 staining result, cell ultrastructure experiment was carried out. Drug-containing serum 0% and 15% for 24 h were added to the cells. e cells were scraped by a cell scraper, fixed with 2.5% glutaraldehyde, washed with 0.1 M phosphate buffer, dehydrated through a graded ethanol series, followed by embedding, and imaged on H-7650 TEM (HITACHI, Japan).
3.6. Western Blot Assay. SMMC-7721 cells were treated with complete culture medium containing different concentrations of medicated serum (0, 5, 10, and 15%) and cisplatin (4 μg/ mL) for 48 h, centrifuged to remove the supernatant, and washed three times with PBS. An appropriate amount of cell lysate containing PMSF was added, and cells were lysed on ice. After quantification by the BCA protein quantification kit, GAPDH was used as an internal reference for Western Blot. Photoshop software, Image J, and GraphPad software were used to statistically calculate the data to analyze the expression of related autophagy proteins such as Beclin1, mTOR, and STAT3 in liver cancer SMMC-7721 cells. e level of protein and phosphorylation expression were measured.

qRT-PCR Assay.
Cells treated with Cisplatin and different concentration ZZXJD-containing serum for 48 h. e total RNA of each group cells was extracted with Trizol (Invitrogen, USA). Use the NanoPhotometer ™ P-Class ultramicro spectrophotometer to measure the light absorption values at 260 nm and 280 nm, and record the ratio of the two to determine the purity of the extracted RNA. e ratio of all test samples is required to be controlled between 1.8 and 2.1 to ensure the purity of the extracted RNA. Use PrimeScript First Strand cDNA Synthesis Kit to reversetranscribe RNA into cDNA according to the instructions. Adopt SYBR premixed ExTaq kit (Takara, Dalian, China); PCR amplification was conducted at 35 cycles of denaturation at 95°C for 30 s, after predenaturation treatment at 95°C for 5 min, annealing treatment 58°C for 30 s and extension at 72°C for 30 s. e temperature at the end was 4°C. GAPDH was used as the internal reference gene to detect the relative expression levels of Beclin1, mTOR, STAT3, and other related genes. e primers of these gene were displayed in Table 2. Each sample was repeated three times to ensure the accuracy of the data. e relative expression levels of these genes were calculated in accordance with 2 −∆∆ct .

Gene Chip.
Gene chip experiments divided into two groups, which were the model group and 20% ZZXJT containing serum as drug group (noted drug and NC). e experiment was repeated three times. e RNA extraction was the same as the RT-PCR extraction. e total RNA extracted was tested by NanoDrop 2000 and Agilent Bioanalyzer 2100. e quality control standards were ermo NanoDrop 2000 1.7< A260/A280 < 2.2, Agilent 2100 Bioanalyzer RIN ≥ 7.0 and 28S/18S > 0.7. e follow-up chip experiment was carried out after the sample was qualified. e signal intensity, principal component, and correlation analysis were based on chip data. Carry out significant difference analysis and statistical test on chip data qualified for quality control testing. Carry out functional and biological pathway analysis on differential genes. GraphPad Prism software, R language software for graphing analysis of related data, was used.
3.9. Tumor Suppression Experiment. BALB/c nude mice were randomly divided into five groups with 10 mice in each group. All nude mice were subcutaneously injected with 2.4 × 10 7 SMMC-7721 cells resuspended in saline in the right axillary. A round bulge under the axillary was considered successfully after three days. Mice had palpable masses in the right axilla, and the tumor volume was about 45-50 mm 3 . Fifty nude mice were randomly divided into model, low of Table 2: Primer sequences of qRT-PCR.

Genes
Sequences TBST was washed again three times, and the electrochemiluminescence ECL kit was used to visualize the protein bands to reflect the expression level of each protein in the tissue. e experiment was repeated three times.
3.11. Statistics. GraphPad Prism 5 was used for statistical analysis, and the data were all expressed as x ± s. One-way analysis of variance, LSD, and SNK-q test were used for statistical analysis of data.

e Effect of ZZXJD on the Proliferation of Liver Cancer SMMC-7721 Cells.
To determine the antitumor effect of ZZXJD on liver cancer cells, the CCK8 assay was used to study the changes in cell viability. ZZXJD with different final concentrations was applied to SMMC-7721 cells for 24 h and 48 h. As shown in Figure 1 and Table 3, compared with the model group, the results of CCK8 at 24 and 48 h showed that 5-20% of ZZXJD had an inhibitory effect on SMMC-7721 cells, and the difference was statistically significant (p < 0.05). 5-17.5% of ZZXJD had the time-and concentration-dependence. e 24hour result showed that 2.5% had an inhibitory effect on SMMC-7721 cells, but the difference was not statistically significant. It is worth noting that 20% ZZXJD has an inhibitory effect on SMMC-7721 cells at 24 h and 48 h (p < 0.05). But the inhibitory effect was less than 17.5%. As shown in Figure 1, after ZZXJD treatment for 24 h and 48 h, the cell viability of SMMC-7721 cells treated with ZZXJD decreased, and the inhibitory effect gradually increased with the increase of concentration and time at a certain concentration.

e Effect of ZZXJD on the Morphology of SMMC-7721
Cells. e Hoechst staining results are shown in Figure 2. Compared with the model group, the number of cells treated with drug decreased. In the meantime, the shape of cell nucleus changed into shrinkable and round, because the chromatin condensed into an irregular crescent shape. e cell nucleus disintegrated into fragments some time. Compared with ZZXJD group, ZZXJD + 3MA group and the cisplatin group had higher mortality of liver cancer cells. AO/EB double-staining distinguished apoptosis cells at different stages (Figure 3). e more cells were green AO nuclear staining in the control group. e cellular nucleus had condensed chromatin in early-stage apoptosis. e latestage apoptosis cells, with increasing doses and asymmetrically localized orange nuclear EB staining, were also observed.
e apoptotic nonviable cells are stained bright orange because of EB into these cells. In Figure 4, cellular structure of ZZXJD group (high dose; Figures 4(b) and 4(c)) was different to model control group (Figure 4(a)). e nuclear membrane of model group was integrated, whereas that of ZZXJD group was ruptured and the chromatin condensed in margin. In ZZXJD group, cell apoptosis and autophagy occurred at the same time, as the autophagosomes in Figure 4(b). Moreover, mitochondria were swelling and vacuolar degeneration.

e Effect of ZZXJD on Tumor Growth.
To explore the role of ZZXJD on tumor-implanted nude mice, the tumor weight and inhibition rate were analyzed. e results are shown in Figure 5 and Table 4. ZZXJD significantly decreased the tumor growth. e tumor inhibition rates of ZZXJD groups were 30.40%, 37.47%, 42.05%, and 42.05%, respectively. With the increase of the dosage, the tumor size gradually decreased, and the tumor inhibition rate gradually increased. e tumor inhibition rate of cisplatin group was 51.33%, and its tumor weighty was lighter than that of ZZXJD group (p < 0.05).

e Effect of ZZXJD on Protein Expression of Liver Cancer
Cells. Because cell apoptosis and autophagy occurred in the SMMC-7721 cells, the further in vivo experiment  Figure 1 shows that different doses of ZZXJD affected cell viability. * Compared with the model group, it is statistically significant (p < 0.05) in 24 h. Δ Compared with the model group, it is statistically significant (p < 0.05) in 48 h.    Regarding the in vitro experiment, these protein expression analyses of SMMC-7721 cell treated with different drug were shown in Figure 7. Compared with the model group, BAX protein difference of ZZXJD group and cisplatin group was statistically significant (p < 0.05). BCL2 protein expression was downregulated in middle-dose ZZXJD group

37.47%
High-dose group 10 0.673 ± 0.19010 * Δ 42.05% Cisplatin group 10 0.565 ± 0.22471 * 51.33% * Compared with the model group, it is statistically significant (p < 0.05), and Δ is statistically significant compared with cisplatin group (p < 0.05). and cisplatin group. In terms of the protein expression of p-AKT compared with the model group, the difference in ZZXJD and cisplatin group was statistically significant (p < 0.05). For great clarity of autophagy role, there were the other two groups added in the further study, which were 3MA (autophagy inhibition) and 3MA + high-dose ZZXJD group. Compared with the model group, mTOR, p-JAK2, and p-STAT3 protein expression in the other six groups were downregulated (p < 0.05). Compared with cisplatin group, p-JAK2 and p-STAT3 protein expression had no statistically significant difference (p > 0.05), except mTOR (p < 0.05).
When the autophagy inhibitor 3 MA was used in combination with the high-dose ZZXJD, the difference in mTOR, p-JAK2, and p-STAT3 protein expression was statistically significant compared with the model group. Compared with cisplatin group, mTOR, p-JAK2, and p-STAT3 protein expression were not statistically significant (Figure 8).

e Effect of ZZXJD on Gene Expression Level of Liver
Cancer Cells

qRT-PCR to Detect Changes in the Level of Genes in
Cells. e liver cancer SMMC-7721 cells treated with drugs were analyzed by qRT-PCR. e results were shown in Figure 9. Compared with the model group, the autophagy and apoptosis-related genes detected in ZZXJD group were significantly changed (p < 0.05). As shown in Figure 9, JAK2 and STAT3 mRNA expression was gradually downregulated with the increase of the dosage of ZZXJD, and the difference was statistically significant compared with the model group and cisplatin group. When the autophagy inhibitor 3MA was used, JAK2 mRNA expression was downregulated (p < 0.05), while STAT3 mRNA was not statistically significant compared with the model group. In 3MA + highdose ZZXJD group, JAK2 and STAT3 mRNA expression was upregulated (p < 0.05), which corresponded with protein expression results. As shown in Figure 9(b), AKT and mTOR Evidence-Based Complementary and Alternative Medicine mRNA expression in each drug group were statistically significant compared with the model control group, and the inhibition effect of ZZXJD was dose-dependent. e analysis of autophagy-related genes LC3, P62, and BECLIN1 on SMMC-7721 cell line was shown in Figure 9(c). Compared with the model group, with the increase of the dose of ZZXJD, the autophagy-related genes LC3 and BECLIN1 were gradually upregulated, while P62 mRNA was downregulated. When SMMC-7721 cells were treated with 3MA (autophagy inhibitor), the expressions of LC3 and BECLIN1 decreased (p < 0.05), while P62 increased significantly (p < 0.01). In 3MA + high-dose ZZXJD group, LC3 and BECLIN1 mRNA increased, which suggested ZZXJD induced cell autophagy. e analysis of apoptosis-related genes in SMMC-7721 cell was shown in Figure 9(d). With the increase of the dose of ZZXJD, the mRNA expression of BAX and BAX/BCL2 gradually increased. In contrast, BCL2 mRNA expression declined compared with the model group (p < 0.05), and the high-dose ZZXJD group was not statistically significant compared with cisplatin. 3MA + highdose ZZXJD group was similar to high-dose ZZXJD group, and BAX/BCL2 ratio was consistent, too.

Gene Chip.
To analyze the whole transcriptome chip information of the collected sample gene chip, the quality of the original data of the chip was usually evaluated at first, and the subsequent information analysis was performed for the data that was qualified for quality control, including significant difference analysis and functional analysis of differential genes [14].
As shown in Figure 10(a), the correlation analysis was accomplished through person correlation coefficients (PCC). PCC of drug and NC groups were both greater than 0.95, indicating that the gene expression trends of samples in the same group were highly similar. PCC between the two groups were significantly lower than PCC of the same groups, indicating large differences between them, which met the criteria for continued analysis. Figure 10(b) was the scatter diagram of this gene chip, showing the distribution of signal intensity between the ZZXJD group and the model group on the rectangular coordinate system plane. e relatively parallel green solid line is the difference reference line, and the reference line is the points in the interval representing the probe groups with no significant changes. e red points outside the interval represent the probe groups that were relatively upregulated in the ZZXJD drug group (noted drug), and the green points represented the probe groups that were relatively upregulated in the model group (noted NC). Figure 10(c) is a chromosome distribution map of differential genes, showing the distribution of differential genes on chromosomes and the specific positions of differential genes on each chromosome. e red in the figure represents the distribution of upregulated genes on 8 Evidence-Based Complementary and Alternative Medicine the chromosomes, and the green represents the distribution of downregulated genes on the chromosomes. Figure 10(d) is a heat map for hierarchical clustering of drug and NC samples with the expression profiles of differential genes screened by |fold change| ≥ 2.0 and FDR < 0.05 as the standard. Compared with the normal model group, STAT3, STAT5B, JAK1, and IL6ST were relatively downregulated in the medication group. e downstream genes HIF1A and BNIP3L of the JAK/STAT signaling pathway were downregulated, and the downstream CASP9 of the AKT/mTOR signaling pathway was upregulated, which was positively correlated with autophagy. e genes BECLIN1 and TP53INP2 were relatively upregulated, while the gene DCTN4 (P62), which is negatively related to autophagy, was relatively downregulated.

Discussion
Tumor is one of the most harmful diseases to human health, and it is also an important cause of the global burden of disease [15]. Excessive proliferation is one of the important malignant behaviors of tumor cells, so cell death is the research focus. Cell death plays an important role in being a stable balance by removing excess cells. Cell death concludes cell apoptosis (type I of programmed cell death, PCD), cell autophagy (type II of PCD), and necrotic programmed death (type III of PCD). Cell apoptosis is a continuous and dynamic process, which is the start of apoptosis, apoptosis body formation, and being engulfed by phagocytes. Cell autophagy is also a dynamic progress, which is the initiation, autophagosome formation, and being degraded in lysosome. Autophagy is a process of lysosome degradation of intracellular components, which is peculiar to eukaryotic cells. Autophagy is induced by external nutrients deficiency, ischemia, or hypoxia. e pressure also comes from cellular metabolic stress, aged or damaged organelles, and misfolded proteins. Autophagy and apoptosis are double-edged swords for cell growth and survival. rough the preliminary RNAseq analysis, combined with the Western blot and qRT-PCR detection of this experiment, the research team found that the prescription ZZXJD inhibited the proliferation of liver cancer cells and was related to the PI3K/Akt/m TOR and JAK2/STAT3 signaling pathways.
PI3K/AKT/mTOR signaling pathway is an important signaling pathways involved in the progress of autophagy and apoptosis. mTOR is a receptor for amino acids and adenosine-triphosphate (ATP). Its activity is essential for the formation and maturation of autophagosomes. e mTOR pathway had confirmed that a variety of tumors had the feature of the pathway overactivation. Inhibiting this signaling pathway can induce cell cycle arrest, apoptosis, and autophagy. Moreover, it suppresses tumor metastasis and drug resistance [16].
e negative regulation of mTOR signaling pathway can activate ULK1 to phosphorylate it, thereby causing the formation of a series of autophagy complexes to induce autophagy. Conversely, inhibiting this pathway can induce autophagy [17]. STAT3 signaling pathway can be activated by cytokines and participates in all steps of the autophagy process, including autophagosome assembly and maturation. Its role is related to the stage of tumor development and the location of STAT3 protein. STAT3 protein with different locations plays different roles in cell's life activity. Autophagy is an important signal pathway for the occurrence and development of tumors and other diseases [6,18]. After tumor-implanted nude mice were treated with different dose of ZZXJD or cisplatin, the cell proliferation was suppressed significantly compared with the model group. Combined with the in vitro SMMC-7721 cell experiment, ZZXJD and cisplatin both downregulated the gene and protein expression of AKT/mTOR and JAK2/STAT3 and regulated the gene expression of apoptosis-and autophagy-related gene expression, such as BAX, BCL-2, and BECLIN1 [19][20][21]. BECLIN1 is the first discovered key factor related to autophagy regulation. BECLIN1 can form a complex with III-type PI3K and mobilize autophagy-related proteins to localize to the proautophagosome. BAX and BCL2 are both important members of the BCL2 family, where BCL2 family plays an apoptotic role mainly by regulating the opening of mitochondrial membrane channels and the flow of proapoptotic substances. BAX is a key protein in the process of cancer apoptosis, which can promote the apoptosis of cancer cells. BCL2 has the opposite effect of BAX. It is a key protective protein for cancer cells and can inhibit cancer cell apoptosis [22]. e combination of BAX and BCL2 can form an apoptotic dimer, promote the release of apoptosis-inducing factors, and cascade with caspase protein to induce apoptosis [23]. BAX and BCL2 belong to BCL2 family and are involved in the mitochondrial apoptosis [24]. BCL2 not only played an important role in apoptosis, but also in autophagy. LC3 is  Figure 9: Gene expression in SMMC-7721 cells (X ± s). * Compared with the model group, it is statistically significant (p < 0.05), and Δ is statistically significant compared with the positive control group (p < 0.05). a marker protein for detecting autophagy and is the first autophagosome membrane protein. In the process of autophagy, LC3 is cleaved by ATG4 with endoproteinase activity to produce LC3-I, LC3-I. Ubiquitin-like reaction interacts with phosphatidylethanolamine (PE) to produce membrane-bound form of LC3-II, which is attached to the autophagosome membrane, and the expression level of LC3-II is positively correlated with autophagy [25][26][27]. While Bcl-2 was upregulated, LC3II/I ratio was decreased in the tumors, indicating inhibited apoptosis and autophagy [28]. P62 is one of the autophagy substrate connexins. It has multiple domains. It plays a role by carrying the protein to be degraded into the autophagosome, and it is continuously degraded by the lysosome during the autophagy process [29,30]. When autophagy is inhibited, p62 can accumulate in cells, and its expression level is negatively correlated with autophagy. In the gene chip assay, P62 gene (DCTN4) expression of ZZXJT group cell was downregulated compared with the untreated cells. is indicated that ZZXJT induced SMMC-7721 cell autophagy. Gene chip results also showed that HIF1A and CCND1 mRNA expression decreased. Including BCL2, they were the downstream gene of JAK/STAT signaling pathway and AKT/mTOR [31][32][33][34], too.

Conclusion
With these findings of the exploration, we conclude that Zhenzhuxiaoji decoction induced SMMC-7721 cells apoptosis and autophagy by JAK/STAT and PI3K/AKT/mTOR signaling pathways. Because the target of Chinese medicine is multitudinous, the study will go on.