Investigating the Effect of Hydrogen-Rich Water on Liver Cell Injury and Liver Cancer by Regulating GP73/ TGF-β Pathway

intervention effect of whether regulated by signaling pathway would provide a for using in ratios. (C1) Hydrogen-rich water increases the content of TGF-β in the supernatant of the LX-2 cell dominant growth group. (C2) Hydrogen-rich water decreases the content of TNF-α in the supernatant of the LX-2 cell dominant growth group. (D1) Hydrogen-rich water increases in equal proportion cell growth the content of TGF-β in the supernatant of the group. (D2) The content of TNF-α in the supernatant of the cell growth group increased with hydrogen-rich water. (E1) The content of TGF-β in the supernatant of the HepG2 cell growth group with the intervention of hydrogen-rich water. (E2) Hydrogen-rich water interferes with the TNF-α content in the supernatant of the HepG2 cell dominant growth group. (F) Hydrogen-rich water interference with the OPN content in the supernatant of the HepG2 cell dominant growth group.


Introduction
Liver cancer is an extremely heterogeneous malignant disease among tumors identi ed to date. Hepatocellular carcinoma (HCC) arises from chronic liver in ammation and brosis and takes a varied course in individual patients up to tumor formation. Risk factors, such as hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections, a atoxin infection, abused alcohol intake, metabolic syndrome, obesity, and diabetes are closely related to the environmental and genetic susceptibilities to HCC [1]. Golgi protein 73(GP73) is a type II Golgi membrane protein, which is a transmembrane glycoprotein. Kladney et al. [2] rst isolated Golgi protein GP73 during a genetic screening from the liver of a patient with adult giant-cell hepatitis (GCH). The expression of GP73 is increased markedly in HCC cells, and the serum levels are predictive of HCC [3][4][5]. Several studies have reported GP73 as a serum marker for HCC [6][7][8]. In patients with acute and chronic hepatitis, cirrhosis, and liver cancer, the level of GP73 is signi cantly increased. TGF-β belongs to the superfamily of transforming growth factor-beta (TGF-β) signaling molecules and is widely expressed across nematodes to mammals. It is synthesized and secreted in an inactive form in all types of cells, and only after activation and binding to the receptor, it exhibits biological activity and pathophysiological effects. Furthermore, TGF-β is a multifunctional cytokine that is regulated via the TGF-β/Smad2 signaling pathway and closely related to the occurrence, invasion, and metastasis of many tumors [9]. The TGF-β signaling pathway interferes with hepatocyte proliferation, and also accelerates HCC progression. Thus, TGF-β has been reported to play both tumor-suppressive and tumor-promoting roles [10].
Hydrogen is a reductive small gaseous molecule that exhibits "antioxidant," "anti-in ammatory," and "anti-apoptotic" effects in vivo [11,12]. In recent years, a large number of studies have found that lowconcentration hydrogen or hydrogen-rich water or hydrogen-saturated physiological saline has a protective effect on many diseases. Some studies have shown that hydrogen-rich water signi cantly reduces the side effects caused by radiotherapy and improves the quality of life of radiotherapy patients.
On the other hand, a few studies have shown that hydrogen-rich water has a certain preventive effect on kidney cancer and liver cancer caused by non-alcoholic steatohepatitis. In this study, we established Nnitrosodiethylamine (DEN)-induced hepatic stellate cell injury model, liver cancer HepG2 cells, and HepG2 and LX-2 co-culture system to observe the intervention effect of hydrogen-rich water and speculate whether its effect is regulated by GP73/TGF-β signaling pathway. These ndings would provide a basis for clinical treatment using hydrogen in liver cancer.
LX-2 cells were cultured in RPMI 1640 (Giboc, New York, USA, NO.C11875500BT) containing 10% fetal bovine serum (FBS) and various concentrations of DEN (10, 25, 50, 80, 100, 160, and 200 μmol/L, respectively) for 16 h. The cell survival was detected by the CCK8. The concentration of LX-2 cells in the logarithmic growth phase was adjusted to 1×104 cells/mL, and a volume of 200 μL was plated/well in a 96-well culture plate. Subsequently, the cells were divided into normal, DEN model, and test groups (including hydrogen-rich water medium group). The normal and DEN model groups did not receive any treatment. The test group was treated with hydrogen-rich water for 16 h, and the cell survival rate was detected by the Cell Counting Kit-8 (CCK-8) method to observe the protective effect of hydrogen-rich water on LX-2 cells. HepG2 cells were cultured with RPMI 1640 containing 10% FBS, the cell density was adjusted to 1×104 cells/mL, and 200 μL/well dispensed in a 96-well culture plate. Then, 1, 2, 3, and 4 number of times hydrogen-rich water and ionized water medium were added for 8 h and 24 h, respectively, following which, the cell viability was detected by CCK-8 assay. The LX-2 and HepG2 cells were co-cultured at the ratios of 1:3, 1:1, and 3:1, respectively. Hydrogen-rich water was added to the coculture system for 24 h, and the cell viability was detected by the CCK-8 method. After the intervention of the above-mentioned cells and hydrogen-rich water, the cell supernatant was collected, and the content of transforming growth factor (TGF-β), tumor necrosis factor-alpha (TNF-α), and osteopontin (OPN) was detected by enzyme-linked immunosorbent assay (ELISA). The above-mentioned cell culture and hydrogen-rich water intervention groups were subjected to total cell RNA extraction and reverse transcription. Real-time PCR was used to quantify GP73, TGF-β, and Smad2 mRNA, and β-actin was used as an internal reference to calculate the relative expression of the target genes using the 2 -△△CT method.
Total protein was extracted from the above cell culture and hydrogen-rich water intervention groups and quanti ed. An equivalent of 20μg total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVD membranes (Millipore, Beijing, China, NO.R8HA7228), blocked, and probed with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with the corresponding secondary antibodies at room temperature. Finally, the immunoreactive bands were visualized using enhanced chemiluminescence (ECL) and images acquired.
All data were analyzed using SPSS16.0 software and expressed as x ±SD. One-way analysis of variance (ANOVA) was used for comparison among multiple groups, and P<0.05 indicated statistical signi cance.
The plots were constructed using GraphPad Prism 8.

The protective effect of hydrogen-rich water on the damage of LX-2 cells induced by DEN
Different concentrations of DEN were used to intervene in LX-2 cells. As the concentration of DEN increased, the cell activity of LX-2 decreased, and the content of TGF-β and TNF-α in the cell supernatant showed an increasing trend. When the concentration of DEN is 200 μmol/L, the cell activity of LX-2 is signi cantly reduced compared to the control group without DEN (Fig. 1A). Also, the contents of TGF-β and TNF-α in the supernatant increased ( Fig. 1B and C). In the presence of different concentrations of DEN in LX-2 cells, the medium prepared with hydrogen-rich water was replaced in the experimental group.
These results showed that when the concentration of DEN was 200 μmol/L, the cell viability of the experimental group was increased signi cantly compared to the model group without hydrogen-rich water (P<0.001) (Fig. 1D). Compared with the model group, the content of TGF-β in the supernatant of the experimental group increased slightly (Fig. 1E), and the content of TNF-α increased signi cantly (Fig. 1F). Therefore, DEN causes damage to LX-2, and after hydrogen-rich water intervention, it exerts a protective effect on DEN-induced cell damage.
Real-time PCR results showed that when 200 μmol/L DEN was used to induce LX-2 cells, the expression of GP73, TGF-β, and Smad2 mRNA was upregulated to varying degrees compared to normal LX-2 cells ( Fig. 2A -C). After changing to a hydrogen-rich medium, GP73, TGF-β, and Smad2 mRNA in the cells was signi cantly downregulated compared to the model group ( Fig. 2A-C). Western blot results showed that after hydrogen-rich water interfered with DEN-induced LX-2 cells, the protein level of GP73 and TGF-β in the cells was decreased compared to the model group, but no signi cant inhibitory effect was noted on the level of Smad2 protein ( Fig. 2D-G). This phenomenon showed that hydrogen-rich water protects DEN from damage by inhibiting the expression of GP73 and TGF-β mRNA and protein in the cells. It is also speculated that DEN induced damage in LX-2 cells, but the damage did not progress to the stage of liver cancer, and hence, hydrogen-rich water did not have a signi cant effect on the protein expression of Smad2 in the cells.

The effect of hydrogen-rich water on HepG2 liver cancer cells
The cell viability data of HepG2 cells treated with hydrogen-rich water culture medium for 8 h showed that the viability of HepG2 cells with three volumes of hydrogen-rich medium decreased signi cantly, indicating an inhibitory effect of hydrogen on HepG2 cells (Fig. 3A). Similarly, after 24 h of HepG2 cell culture medium prepared with hydrogen-enriched water for different durations, the activity of the cells given hydrogen-enriched medium three times decreased signi cantly compared to the control group without hydrogen-enriched water medium (P<0.05) (Fig. 3B). HepG2 intervened with hydrogen-rich medium three times and treated for 24 h showed that when the number of HepG2 cells was 5000 cells/well, hydrogen gas signi cantly inhibited the activity of liver cancer cells as compared to the control group without hydrogen-enriched water (P<0.01), and a maximum inhibitory effect was also observed (Fig. 3C). The determination of TGF-β and TNF-α in the cell supernatant showed increased secretion of TGF-β after adding hydrogen-rich medium for different times. When the hydrogen-rich medium was given three times simultaneously, the level of TNF-α increased in the supernatant of the cells, which in turn inhibited the proliferation of liver cancer cells (Fig. 3D and E). Thus, the addition of hydrogen-rich medium three times was preferred as the optimal conditions to intervene in HepG2 cells. The results showed that hydrogen intervention increases the content of TGF-β and TNF-α in the supernatant of HepG2 cells, which is speculated to elevate TGF-β--inhibited degradation of extracellular matrix and prevent the migration and invasion of liver cancer ( Fig. 3F and G).
The intervention of HepG2 cells with a hydrogen-rich medium inhibited the expression of GP73 and Smad2 mRNA in the cells (Fig. 4A and C) and increased the expression of TGF-β mRNA (Fig. 4B). Western blot showed that after intervention with hydrogen-rich medium, the expression of GP73 protein was downregulated in the cells (Fig. 4E), and that of TGF-β protein was upregulated (Fig. 4F), but no signi cant change was detected in Smad2 protein. (Fig. 4G). Thus, it was speculated that hydrogen not only passes through the GP73/TGF-β pathway but might also inhibit the proliferation of liver cancer cells via other pathways.

Study on the effect of hydrogen-rich water on the co-culture system of LX-2 and HepG2
The co-cultivating of LX-2 and HepG2 cells at different ratios revealed that the growth rate of LX-2 cells was signi cantly lower than that of HepG2 cells. When the two types of cells were co-cultured for 4 h, HepG2 cells were found to fuse with LX-2 cells, as assessed by microscopy. With prolonged culture time, the fusion effect became pronounced, indicating that HepG2 cells are highly invasive (Fig. 5).
The hydrogen-rich medium was added to the co-culture system of LX-2 and HepG2 cells (1:1) in different proportions. Microscopic analysis revealed that hydrogen intervention exerts a signi cant inhibitory effect on cell proliferation (Fig. 6A, 6B). This phenomenon is consistent with the inhibitory effect of the previous hydrogen-rich medium on different numbers of HepG2. When LX-2 cells are dominant in the co-culture system, hydrogen increases the secretion of TGF-β in the cell supernatant (Fig. 6C1), thereby promoting the proliferation of normal liver cells in the co-culture system to combat the invasion of normal cells by liver cancer cells. Furthermore, in the co-culture system, with the increase in the number of HepG2 cells in hepatocytes, hydrogen gas inhibits the secretion of TGF-β and prevents excessive secretion of TGF-β from causing excessive proliferation of liver cancer cells (Fig. 6D1). When the HCC cells are dominant in the co-culture system, although the hydrogen-rich medium does not inhibit the secretion of TGF-β after the intervention, the level of TNF-α is elevated (Fig. 6C2, D2, and E2), and the in ammatory factors kill the tumor cells. In order to further explore the reasons for the increase in TGF-β content, we also tested the OPN content in the supernatant of the co-culture system and found that a hydrogen-rich medium effectively inhibits the secretion of OPN (Fig. 6F). The secreted TGF-β cleaves OPN and prevents it from promoting tumor cell growth .

Discussion
DEN is a carcinogenic drug. In this study, we used DEN to interfere with LX-2 normal liver cells to establish a cell damage model. The experimental results showed that as the concentration of DEN increases, the cell activity of LX-2 gradually decreases, and the content of TGF-β and TNF-α in the cell supernatant increases. When the concentration of DEN was 200 μmol/L, the activity of LX-2 cells was signi cantly reduced, suggesting that DEN damages the normal liver cells. After different concentrations of DEN were used to induce cell damage, a hydrogen-rich medium was used for intervention, and it was found that the activity of LX-2 cells increased, indicating a protective effect of hydrogen-rich water on the DEN-induced cell damage. In addition, compared to normal LX-2 cells, the expression levels of GP73, TGF-β, and Smad2 mRNA and protein in DEN-injured LX-2 cells increased but decreased after intervention with hydrogen-rich water. However, the level of Smad2 protein increased continually, indicating that hydrogenrich water protects LX-2 cells from DEN injury by downregulating the expression of GP73 and TGF-β. Thus, it is inferred that LX-2 cells stimulated by DEN are damaged but have not progressed to the stage of liver cancer, and hence, hydrogen-rich water has no obvious effect on the protein expression of Smad2 in the cells.
In the experiment of DEN on LX-2 cells, TGF-β and TNF-α in the supernatant of LX-2 cells increased slightly, and the expression of GP73, TGF-β, and Smad2 mRNA was upregulated, indicating that LX-2 cells appeared to be certain. After the intervention of hydrogen-rich water, the mRNA and protein expression of GP73 and TGF-β in the injured LX-2 cells was downregulated, indicating a protective effect of hydrogenrich water. The cells cannot progress to liver cancer cells, and hence, cannot signi cantly inhibit the expression of Smad2 mRNA and protein. In the co-culture system of liver cancer cells and LX-2, the cell morphology improved after the intervention of hydrogen-rich water; also, inhibition of cell proliferation was observed.
Hydrogen-rich water exerts an inhibitory effect on the proliferation of liver cancer cells. When the number of liver cancer cells reach a certain level, the inhibitory effect of hydrogen-rich water weakens. When liver cancer cells interfere with hydrogen-rich water, hydrogen-rich water inhibits the proliferation of liver cancer cells by signi cantly inhibiting the upregulation of GP73, TGF-β, and Smad2 mRNA expression and the upregulation of GP73 protein level. Among these, the increased mRNA and protein expression of TGF-β indicates that when the number of liver cancer cells peak, hydrogen-rich water plays a role in the further growth of liver cancer cells via upregulated expression of TGF-β.
In summary, the inhibitory effect of hydrogen-rich water on HepG2 liver cancer cells and the protection of normal hepatic stellate cells are achieved by regulating the GP73/TGF-β/Smad2 signaling pathway, thereby demonstrating the effects of hydrogen-rich water on different cells at various stages. Thus, these ndings provide a scienti c basis for an in-depth investigation on the role of hydrogen-rich water.

Conclusion
Hydrogen-rich water has a protective effect on liver cell damage and an inhibitory effect on liver cancer cells produced by regulating the GP73/TGF-β signaling path.