Effects Of Total Glycosides Of Cistanche Deserticola On Proliferation, Apoptosis And Expression Of Wnt/β-Catenin Signaling Pathway-related Protein Of HepG2 Cells

FENG Duo1,2, WANG Jing2 , JIANG Yong-jun3 , ZHOU Shi-qi1 , DUAN Hao1 ,GUO Yu1 , ZHAO Jian1 , YAN Wen-jie1,* 

（1.Beijing Key Laboratory of Bioactive Substances and Functional Food, College of Biochemical Engineering, Beijing Union University, Beijing 100023, China; 2. Institute of Food and Nutrition Development, Ministry of Agriculture and Rural Affairs, Beijing 100081, China; 3. Inner Mongolia Sankou Biotechnology Co., Ltd., Ordos, Inner Mongolia 017000, China） 

Abstract: To explore the inhibitory effect of total Glycosides of Cistanche deserticola (TG) on HepG2 cells and its mechanism. In this paper, different concentrations (0, 3.5, 10.5, 21, 31.5, 42 μg/mL) of TG were treated for 24 h on HepG2 liver cancer cells, and the viability of HepG2 cells was detected using CCK8 assay. Hoechst33342/PI double staining method and Annexin V-FITC / PI were used to detect HepG2 cell apoptosis. The phenomenon of cell migration was detected by cell migration assay. Meanwhile, cell cycle progression changes were detected by flow cytometry. And the expression of α-fetoprotein(AFP), β-catenin, Dishevelled (Dsh), and GSK-3β was detected by western blot. The results showed that TG could reduce the proliferation of HepG2 cells in a concentration-dependent manner, with only 31.04% cell viability when TG was 42 μg/mL. In addition, TG could damage the cell structure and induce cell apoptosis, and the apoptosis rate could be as high as 32.44% by AV/PI detection.Moreover, TG could also promote cell necrosis, and limit cell migration. There were significant differences between the treated group and the control group. Meanwhile, a high concentration of TG could arrest HepG2 cells in the G2/M phase. Finally, compared with the control group, the relative expression of β-catenin and Dsh decreased, while GSK-3β increased in the TG-treated groups. In a conclusion, TG could inhibit HepG2 cell growth by affecting cell cycle progression, promoting apoptosis, and limiting cell migration. The mechanism may be through the Wnt/β-catenin signaling pathway, which activated GSK-3β to degrade β-catenin to achieve liver cancer inhibition. 

Keywords: Cistanche deserticola ; total glycosides; HepG2 cell; anti-hepatoma; Wnt/β-Catenin signal pathway

Cistanche deserticola ma belongs to a medicinal and food homologous substance and is a perennial herb parasitic plant of the Orobaceae. It has the effects of antioxidation, anti-fatigue, anti-aging, anti-tumor, liver protection, endocrine regulation, improving memory ability, and anti-osteoporosis. It is known as "Desert Ginseng". After confirming that deserticola deserticola has become a medicinal and food homologous substance in 2018, it has attracted much attention. It can not only play the clinical application value of Chinese herbal medicine but also affect the nutritional intervention effect of food. It also shows great potential in the development of new natural anti-liver cancer products [2-3]. The nutritional research and functional effects of deserticola deserticola foods are becoming increasingly in-depth [4]. 

Desert living cistanche

In recent years, it has been reported that Cistanche deserticola has an anti-tumor effect. Ye et al. [5] have found that echinacea extracted from Cistanche deserticola salt can inhibit the proliferation of HepG2 cells by reducing the expression of TREM2 and blocking the PI3K/AKT signal pathway; Some scholars have found that phenylethanoid glycosides from Cistanche deserticola can inhibit the proliferation of HepG2 cells [6]; Echinoside has a certain inhibitory effect on the proliferation of renal cancer 786-O cells [7] and SW480 colon cancer cells [8]; In another study, phenylethanoid glycosides from Cistanche deserticola have been shown to reduce liver damage in H22 tumor-bearing mice, and improve immune function by reducing AFP levels, thereby adversely affecting tumor growth [9]. Both Cistanche deserticola decoction [10] and polysaccharide [11] can be passed through Wnt/ β- Catenin signaling pathway improves the clinical symptoms of Parkinson's rats and plays a neuroprotective role. In addition, studies have found that echinacea can inhibit Wnt by/ β- Catenin signaling pathway plays an anti-breast cancer role [12]; In addition, it can also reduce THP-1 cells in human acute leukemia β- Catenin protein expression [13]. All along, Wnt/ β- Catenin signaling pathway has received considerable attention [14]. It is reported that it can be observed in 20% - 35% of HCC cases [15] β- Activation of Catenin. There is increasing evidence that the Wnt/ β- Catenin signaling pathway plays an important role in the occurrence and development of liver cancer [16], but whether total glycosides of Cistanche deserticola regulate Wnt/ β- There are few reports on the anti-hepatoma effect of the Catenin signaling pathway. The total glycosides of Cistanche deserticola extract, including phenylethanoid glycosides and other glycosides [17], were studied in this experiment by studying the proliferation, apoptosis, migration, and related protein expression levels of HepG2 cells induced by Total Glycosides of Cistanche deserticola (TG), to explore the mechanism of TG inhibiting liver cancer, and to explain the potential mechanism of TGs against liver cancer, To provide the certain scientific basis for further enriching the clinical application of Cistanche deserticola in the field of liver cancer.

Phenylethanol glycoside is the main active component of Cistanche deserticola

1. Material and Methods

1.1 Materials and Instruments

HepG2 (Human Hepatocellular Carcinomas) cells were purchased from the Cell Center of the Institute of Basic Medicine, Peking Union Medical University, China. The total glycosides of Cistanche extract powder (mass fraction ≥ 75%, combined with echinacoside and poolside, provided by Inner Mongolia Sankou Biotechnology Co., Ltd.) were dissolved in DMEM medium to corresponding concentrations of TG for subsequent experiments. 

Cistanche extract powder

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Fetal bovine serum was purchased from Gibco, USA; Antibodies (penicillin-streptomycin gentamicin mixed solution), DMEM-H medium, cell apoptosis and cell cycle detection kit, Annexin V-FITC/dye solution, and apoptosis Hoechst 33342/PI dye solution were purchased from Genview; The 0.25% trypsin and BCA protein concentration determination kit were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd; Rabbit anti β- Catenin antibodies were purchased from ABclonal; Rabbit anti-Dsh (ADAR1) antibody was purchased from Bios; Rabbit anti GSK3 β Antibodies and rabbit anti-AFP antibodies were purchased from Affinity. CO2 cell incubator (Innova CO-170), low-temperature high-speed desktop centrifuge (3K15, Sigma, Germany), multifunctional enzyme labeling instrument (INFINITE M NANO TECAN), fluorescence microscope (C-SHG1, Nikon, Japan), flow cytometry (FACSCALIBUR, BD, USA), electrophoresis device (EN027015, BIO-RAD, USA), membrane transfer device (043BR57802, BIO-RAD, USA). 

1.2 Experimental method

1.2.1 Cell passage

HepG2 cells were cultured in a DMEM complete medium containing 10% fetal bovine serum and 1% triple antibodies (100 U/mL penicillin, 100 mg/mL streptomycin, and gentamicin) in a 5% CO2 cell incubator at 37 ℃. After the cells grew to a confluence of 80% to 90%, they were digested with 0.25% trypsin (- EDTA) and subcultured at a ratio of 1:2 to 4. After 2-3 days of cell growth, logarithmic growth phase cells were taken for a subculture or subsequent experiments

1.2.2 Effect of TG on the morphology of HepG2 cells

HepG2 cells of the logarithmic growth phase were taken and inoculated in a 24-well plate with a cell concentration of 5 per well × 104 pieces/mL, after the cells adhere to the wall, add TG to the final concentration of 0, 3.5, 10.5, 21, 31.5, 42 μ G/mL, 0.5 mL per well, cultured for 24 hours, and observed the cell morphology of each group under a light microscope.

1.2.3 Effect of TG on the proliferation of HepG2 cells

Take HepG2 cells at the logarithmic growth stage and adjust the cell concentration to 1 × 104 pieces/well, inoculated in 96 well plates, adding 100 pieces per well μ 50. Incubate in a cell incubator. After the cells adhere to the wall, they are treated with drugs according to 1.2.2 above, and each group has six reperforations, cultured for 24 hours. The cell survival rate was measured using the CCK8 method, and the absorbance value (OD) was measured at a wavelength of 450 nm. The cell survival rate and cell inhibition rate were calculated using the following formula.

In the formula, the blank group only contains CCK8 liquid, while the control group contains 0 μ G/mL treatment group, experimental group: 3.5, 10.5, 21, 31.5, 42 μ G/mL treatment group.

1.2.4 Effect of TG on HepG2 cell cycle

Take logarithmic growth phase cells and press 5 × A cell density of 105 cells per well was inoculated into a six-well plate, and a system of 2 mL per well contained 5 × 105 cells were placed in a culture box and subjected to drug treatment as described in 1.2.2 above. After the culture, subsequent operations were performed according to the kit instructions.

1.2.5 Effect of TG on the Migration of HepG2 Cells:

 Select logarithmic growth phase cells and lay them on a six-well plate. A system of 2 mL per well containing 6 × 105 cells was removed when the cell adhesion and cell aggregation were above 90%. Conduct drug treatment according to 1.2.2 above. At 0 h, randomly select 5 fields of view under the microscope for photographs, place them in the incubator for further cultivation for 24 hours, take photos under the optical microscope, use Image J software to analyze the scratch area of cells, and calculate the migration rate according to the following formula (2) [18].

1.2.6 Hoechst 33342/PI double staining method for detecting apoptosis

Select logarithmic growth phase cells and lay them on a 24-well plate, with a 0.5 mL system containing 3 × 104 cells were treated with drugs according to 1.2.2 above, and 1 mL of cell staining buffer was added, followed by 5 μ L Hoechst dye and 5 μ LPI dye solution for staining. Incubate in dark at 37 ℃ for 7-10min, take out a 24-hole plate, moisten it with PBS, and take photos with a fluorescence microscope for observation.

1.2.7 Detection of HepG2 cell apoptosis by Annexin V-FITC/PI double staining

Select logarithmic growth phase cells and lay them on a six-well plate, with a system of 2 mL per well containing 2 × 105 cells were treated with drugs according to 1.2.2 above, and subsequent operations were performed according to the kit instructions, adding 5 μ L Annexin V-FITC and 5 μ L PI staining solution was incubated at 4 ℃ in dark and then placed on flow cytometry for apoptosis detection.

1.2.8 Effect of TG on protein expression in HepG2 cells

Press 2 × 106 cells/cell were treated with drugs according to 1.2.2 above, using 80 μ L lysate (RIPA: PMSF=100:1) lyses proteins, and then using SDS-PAGE electrophoresis to separate each protein sample (50 samples per well μ g) Complete the transfer of protein to the PVDF membrane in an ice bath, using 1 × Clean the tape with TBST buffer solution, slowly seal the BSA shaking table at room temperature for 1 hour, and add an antibody (AFP, GSK3) in proportion- β、β- Catenin, Dsh), incubate at 4 ℃ overnight, and then incubate the PVDF membrane with the diluted secondary antibody at room temperature for 1-2 hours. GAPDH is used as an internal reference. After developing and fixing the PVDF membrane, observe the changes in target protein expression [6].

1.3 Data analysis

ANONA single factor significance analysis was conducted using SPSS 25.0 statistical software to express; The experimental data of flow cytometry were analyzed using Flowjo; Use Graphpad Prism 8.0.2 for drawing. P<0.05 indicates a statistically significant difference, while P<0.01 indicates a highly significant difference.

2. Results and Analysis

2.1 Effect of TG on the morphology of HepG2 cells

figure 1  hepg2 cells treated with different concentrations of tg for 24 h (200x)

During the process of apoptosis, first of all, its volume will gradually decrease and deform, and then adherent cells will slowly undergo processes such as shrinking, rounding, shedding, and intracellular chromosome pyknosis. Some nuclei will undergo breakage, marginalization, and the formation of apoptotic vesicles. As observed in Figure 1, 3.5, and 10.5 μ When HepG2 cells were treated with g/mL TG, the cell volume gradually decreased; And then visually observe 21 μ At g/mL, the nucleus began to shrink and the volume became smaller; thirty-one point five μ At g/mL, the cells were accompanied by floating and cell debris; forty-two μ At g/mL, the cell membrane completely ruptures, the cells undergo lysis, necrosis occurs, and the boundary is unclear. The cells are in a state of impending collapse and death. 

2.2 Effect of TG on the proliferation of HepG2 cells

Figure 2   Effect of TG on the proliferation of HepG2 cells

Note: * represents the same as 0 μ The difference was significant at g/mL (P<0.05), and * * indicates a significant difference (P<0.01).

As can be seen from Figure 2, after 24 hours of treatment with different concentrations of TG, the proliferation of HepG2 cells is restricted to varying degrees. Compared with the cell survival rate in the control group, the cell survival rate in the treatment group shows a downward trend with increasing concentration, which is 96.95%, 92.59%, 92.78%, 77.24%, 31.04%, and the concentration is 3.5, 10.5, and 21, respectively μ There was no significant difference in g/mL and the decrease was small, while 31.5% μ G/mL and 42 μ At g/mL, there was a significant difference (P<0.01), with an IC50 of 37.77 after TG treatment μ g/mL。 CCK8 test results indicate that when TG concentration is 21 μ When the concentration is above g/mL, the inhibitory effect on HepG2 cells is better.

2.3 Effect of TG on HepG2 cell cycle

Figure 3   Effects of different concentrations of TG on the cell cycle of HepG2 cells

As shown in Figure 3, after 24 hours of treatment with different concentrations of TG, it was found that as the concentration increased, the G0/G1 phase gradually decreased, accounting for 59.9%, 56.7%, 56%, 54%, 33.4%, and 29.1%, respectively, with a significant decrease; The proportion of phase S was 14.3%, 15.8%, 16.8%, 18.7%, 25.1%, and 27.6%, respectively, with varying degrees of increase; 3.5-21 compared to the control group (23.88%) μ At g/mL, the G2/M phase has little change, accounting for 25.85%, 24.92%, and 25.33%, respectively. When TG is 31.5 μ G/mL and 42 μ At g/mL, it increased significantly, 37.89% and 39.42%, respectively. This test found that TG at low concentrations (3.5-21 μ G/mL), does not inhibit cell division and proliferation in the main way that affects the cell cycle, and at high concentrations (31.5 μ G/mL and 42 μ G/mL) can induce cell arrest in the S phase and G2/M phase, thereby inhibiting cell proliferation. At the same time, when TG is 31.5 μ G/mL and 42 μ At g/mL, subG1 also increased, which is consistent with the research by Sun Qian et al. [19] that artemisinin derivatives can increase the subG1 peak of HepG2 cells at high concentrations. He also found that with the increase in artemisinin concentration, the proportion of G2/M phase cells increases. The results of the cell cycle suggest that TG may promote the inhibition of cell cycle progression, thereby inducing cell apoptosis.

2.4 Effect of TG on HepG2 cell migration

Figure 4   Effects of different concentrations of TG on HepG2 cell migration

Note: * represents the same as 0 μ The difference was significant at g/mL (P<0.05), and * * indicates a significant difference (P<0.01).

Cancer cell metastasis is one of the keys to cancer treatment, and cancer recurrence is also closely related to it. In order to investigate whether TG can inhibit the migration of HepG2 cells, a scratch method was used to observe cell migration. As shown in Figure 4, with the increase of TG concentration, the 24-hour mobility gradually decreases, and the obvious inhibition effect is. The mobility is 14.82%, 13.42%, 12.66%, 10.11%, 8.46%, 7.24%, 21 μ G/mL showed significant differences (P<0.05), 31.5 μ G/mL and 42 μ G/mL showed a significant difference (P<0.01). The results of this experiment indicate that TG can inhibit the migration of liver cancer cells, thereby limiting the metastasis of cancer to other tissues.

2.5 Hoechst 33342/PI double staining method for detecting apoptosis

Figure 5   Effects of different concentrations of TG on Hoechst 33342/PI in HepG2 cells

Nuclear staining was performed with Hoechst 33342 to detect chromatin condensation [20]. Normal cells showed low blue, apoptotic cells showed high blue/low red and necrotic cells showed low blue/high red. As can be seen from Figure 5, compared with the control group, in the TG treatment group, chromatin concentration and nuclear fragmentation were observed. At the same time, when TG was at 3.5-21, it was observed that μ In the range of g/mL, blue fluorescence gradually increased, suggesting that TG mainly inhibits cell proliferation through apoptosis at low concentrations; And above 21 μ At g/mL, red fluorescence increases, suggesting that TG mainly damages and destroys the cell structure to kill cells at higher concentrations. The results of this experiment indicate that treatment with TG can induce apoptosis and necrosis of HepG2 cells.

2.6 Annexin V-FITC/PI double staining to detect the effect of TG on HepG2 cell apoptosis

Figure 6   Effect of TG on AV/ PI double staining of HepG2 cells

Note: * represents the same as 0 μ The difference was significant at g/mL (P<0.05), and * * indicates a significant difference (P<0.01).

To further determine the apoptosis-inducing effect of TG on HepG2 cells, we used Annexin V-FITC/PI double staining method to stain HepG2 cells. As shown in Figure 6, with the increase of concentration, the apoptosis rate shows an upward trend, 0, 3.5, 21, 41 μ The apoptotic rates of g/mL were 5.63%, 7.65%, 10.93%, and 32.44%, respectively, in a dose-dependent manner. The results showed that TG can induce apoptosis by destroying the membrane integrity of HepG2 cells. This result is consistent with the AV/PI staining results of HepG2 cells discussed by Qi Xinxin et al. [6].

2.7 Effect of TG on protein expression in HepG2 cells

Figure 7   Protein expression in HepG2 cells treated with different concentrations of TG

Note: * represents the same as 0 μ The difference was significant at g/mL (P<0.05), and * * indicates a significant difference (P<0.01).

Alpha-fetoprotein (AFP) is used as a serum marker for the diagnosis and efficacy testing of primary liver cancer [21]. As can be seen from Figure 7, compared with the control group, the expression level of AFP in the treatment group has decreased, suggesting that TG can inhibit the occurrence of liver cancer to some extent, but the probability of reversing liver cancer is very small. In addition, Dsh β- The level of Catenin decreased linearly with the increase of drug concentration; GSK-3 β Gradually increased, but not in a dose-dependent manner. Therefore, it is speculated that TG can regulate Dsh β- Catenin and GSK-3 β Relative expression, via Wnt/ β- Catenin classical signaling pathway to induce apoptosis in HepG2 cells.

3 .Discussion and Conclusion

Liver cancer is one of the most common malignant tumors in China and also ranks among the top three diseases with mortality [22]. The main treatment methods for liver cancer include surgical resection, liver transplantation, chemoradiotherapy, and immunotherapy [23-25]. It is reported that in 2016, the incidence rate of liver cancer in China was 9.57%, and the mortality rate was as high as 13.92% [26]. "Due to the nonspecific symptoms of early liver cancer, once symptoms occur, they are mostly in the middle and late stages. Therefore, people are more active in using traditional Chinese medicine to treat liver cancer [27]."People believe that natural compounds have the least molecular toxicity and have proven beneficial for the treatment of liver cancer, so they are actively screening new natural compounds for anti-liver cancer. 

Total glycosides of Cistanche deserticola can inhibit liver cancer cells at the early stage of liver cancer

In recent years, the multiple effects of Cistanche deserticola have attracted much attention and gradually become one of the hot spots in the development of healthy food and drug research. Cell morphology and CCK-8 experiments have confirmed that a certain concentration of TG can destroy cell morphology and inhibit the growth and proliferation of HepG2 cells, and the concentration is in direct proportion to the cell inhibition rate.

The cell cycle refers to the entire process that a cell undergoes from the completion of one mitosis to the end of the next division. It is divided into two stages: interphase and division. Flow cytometry uses fluorescent dyes to distinguish G0/G1 phase, S phase, and G2/M phase. Through cell cycle detection, it is found that after treatment with different concentrations of TG, it is found that at high concentrations of TG (31.5 μ G/mL and 42 μ G/mL) can block cells from being in the G2/M phase and affect the activity of HepG2 cells, resulting in cytotoxicity, thereby inhibiting cell proliferation. During the progression of cancer, cancer cells from primary tumors invade adjacent normal tissues, metastasize to distant sites, and form new colonies. It is estimated that a total of 90% of cancer-related mortality is due to metastasis [28]. The Wnt signaling pathway is involved in the process of epithelial-to-mesenchymal cell transformation and mesenchymal-to-epithelial cell transformation to promote cancer metastasis. In hepatocellular carcinoma (HCC), high levels of β- Catenin is associated with enhanced metastasis and poor prognosis [29]. In this study, it was found that the migration rate of HepG2 cells treated with TG for 24 hours gradually decreased with increasing concentration, in a concentration-dependent manner. This indicates that TG can control the migration of cancer cells and prevent the further spread of cancer, possibly through the reduction of the Wnt signaling pathway β- Expression of Catenin.In general, the affected nuclei appear smaller; Some chromatin concentrates or aggregates at the periphery, while others have typical characteristics such as fragmentation of nuclear chromatin and formation of apoptotic bodies [30-31]. Hoechst 33342 is a blue fluorescent dye that can penetrate the cell membrane and has low toxicity to cells. Some studies have found that morphological characteristics of apoptotic cells, including nuclear coagulation and fragmentation, have been observed in HepG2 cells stained with Hoechst 33342 [32]. In this experiment, it was found that after 24 hours of TG treatment, the proportion of cells with chromatin coagulation and fluorescent nuclear fragments increased in a concentration-dependent manner, which is consistent with the results of the study by Yang et al. [33] on gingerol induced apoptosis of HepG2 cells. Using Annexin V-FITC/PI double staining method to detect HepG2 cells treated with TG for 24 hours, it was found that the apoptosis rate of cells gradually increased with the increase of concentration.

Total glycosides of Cistanche deserticol

Dsh、 β- Catenin and GSK-3 β Is a typical Wnt/ β- Disheveled (Dvl/Dsh) is an important regulator in the Catenin signaling pathway, which is a key protein in the Wnt signaling pathway and can transmit Wnt signals to downstream effectors [34]. In the classical Wnt signaling pathway, Dsh is summoned to the cell membrane by the receptor Frizzled, which acts as a proto-oncogene and is associated with the somatic axis inhibitory factor (Axin), the adenomatous polyposis coli gene (APC), and GSK-3 β Binding to form a degradation complex that inhibits GSK-3 β yes β- The phosphorylation degradation of Catenin causes β- Catenin enters the nucleus and functions [34-36]. β- Catenin is mainly expressed in the cell membrane of normal cells and is related to cell adhesion. It also participates in the information transmission of Wnt signaling pathways, regulating cell proliferation and differentiation. When the Wnt signaling pathway is abnormally activated, β- The redistribution of Catenin may be one of the reasons for tumor occurrence [37]. Reportedly, it can be observed in 20% - 35% of HCC cases [15] β- Activation of Catenin. At the same time, it is also possible to suppress Wnt by/ β- Catenin signaling pathway activates autophagy to inhibit the proliferation of HepG2 cells [38]. This study found that after TG treatment, HepG2 cells, Dsh β- The level of Catenin decreased linearly with the increase of drug concentration; GSK-3 β Gradually increased, but not in a dose-dependent manner. Therefore, it can be inferred that TG passes Wnt/ β- Catenin signaling pathway to induce apoptosis in HepG2 cells.

The most widely used biomarkers of primary liver cancer worldwide are α- Alpha-fetoprotein (AFP) [39]. AFP plays an important role in early monitoring, pathological classification, treatment selection, and prognosis of patients with primary 9 liver cancer. Regardless of the treatment method, when the AFP level is higher than 400 ng/mL, it will lead to poor survival [40]. By detecting the expression level of AFP protein in cells treated with TG, it was found that TG can effectively reduce the expression of AFP, indicating that TG can play a role in inhibiting liver cancer cells at the early stage of liver cancer.

Cistanche tea

In summary, total glycosides of Cistanche deserticola inhibit the proliferation and growth of HepG2 cells by influencing cell cycle progression, destroying cell structure, promoting apoptosis, and limiting cell migration. The mechanism of action may be through Wnt/ β- Catenin signal pathway, activating GSK-3 β Degradation β- Catenin can inhibit liver cancer, but further research is needed. In the future, experts and scholars can deeply explore the application of Cistanche deserticola in Wnt/ β- The upstream and downstream protein molecules of the Catenin signaling pathway are used to verify the target genes and further explore the mechanism of action of total glycosides of Cistanche deserticola against HepG2 cells. In addition, as a medicinal and food homologous substance, cistanche deserticola can be consumed in daily diet to provide both prevention and treatment, and also provide a certain theoretical basis for practicing the Great Food Outlook, promoting the construction of a "Healthy China", and improving the health level of the people.

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