In recent years, studies have shown that SB and HD are commonly used in various cancers such as prostate cancer, ovarian cancer, liver cancer, gastric cancer, and colorectal cancer, etc [18,19,20,21,22,23]. The mechanism mainly includes multiple signaling pathways such as PI3K/Akt/mTOR, AMPK, P53, etc., which inhibit the proliferation, migration and invasion of tumor cells, promote tumor cell apoptosis, and thus achieve the goal of inhibiting tumor growth [18,19,20,21,22,23]. In addition, they can also enhance the body’s immune function and enhance the body’s resistance to tumors [24]. As a natural medicine, they have the potential to be combined with current chemotherapy to improve the therapeutic effect of existing drugs and exhibit good application prospects. In this study, we first evaluate the synergistic effects of SB-HD and DDP on OC, it was found combination treatment obviously inhibited the colony formation, promoted cell cycle arrest and cell apoptosis, inhibited the proliferation of OC cells in vitro and in vivo, and explored its possible mechanisms which is through modulating oxidative stress via NRF2-FTH1 autophagic degradation pathway. The schematic diagram is shown in Fig. 8.

As is well known, the clinical treatment of OC often uses paclitaxel combined with such as carboplatin or DDP which is a combination chemotherapy regimen at present [25]. Although the short-term effect in preventing tumor growth, killing or eliminating tumor cells is optimistic, it cannot be ignored the adverse reactions and drug resistance. Like DDP, it is one of the most effective anticancer drugs and is commonly used to treat advanced OC, while it may occur kidney injury, liver injury, digestive tract injury, some potential neurotoxicity and muscle toxicity caused by long-term use or at high dose [26]. Therefore, in our experiment, we evaluated organ damage in the heart, liver, spleen, lungs, and kidneys of mice, examined the liver, kidney and spleen indices, our results proved that SB-HD reduces the effect of DDP on the kidney damage in OC bearing mice, and has a slight immune protective effect by increasing spleen indices. We also carried out measuring the possible hepatotoxicity and nephrotoxicity of the combination of the two drugs by detecting biochemical indicators ALT, AST, BUN and Cre, which shows that combination treatment markedly reduced the levels of serum ALT, AST, BUN and Cre compared to the DDP alone. Compared with the weight loss of mice in the DDP treatment group, the combination treatment did not affect the weight change, indicating that both SB-HD monotherapy and combination therapy have good safety.

Cancer metastasis is one of the biological characteristics of malignant tumors, which leads to the failure of surgery, radiotherapy and chemotherapy, as well as the death of patients [27]. Cancer cells interact with host cells and affect their microenvironment for survival. Epithelial-mesenchymal transition (EMT) is a process in which tumor cells acquire the ability of migration and invasion due to the loss of epithelial phenotype and the dramatic change of epithelial layer [28]. This process is the main step to promote the metastasis of tumor cells. Here, compared with other types of solid tumors, OC is more likely to metastasize. OC cells mostly grow on the surface of the ovary in the abdominal cavity and are easy to adhere to adjacent tissues. In addition, OC is often accompanied by cancerous ascites, so intra-abdominal contact implantation is the most common mode of metastasis in patients with OC. Most OC patients die from advanced diseases, accompanied by peritoneal metastasis and poor prognosis. Therefore, in this study, we investigated the inhibiting effects of SB-HD alone or in combination with DDP on migration and invasion of OC SKOV3 and OVCAR3 cells, and our results confirmed inhibiting the migration and invasion role in a dose-dependent manner in vitro by wound healing assay, transwell cell migration assay and transwell cell invasion assay. Moreover, it is noteworthy that the expression of p62 is significantly elevated in tumor tissues treated with DDP alone in western blot analysis. It has been reported that p62 as an oncotarget mediates cisplatin resistance and high p62 cytoplasmic expression was shown to be associated with poor prognosis [29, 30]. Besides, Lu et al. found that p62 knockout inhibits migration and invasion of hepatocellular carcinoma [31], high expression of p62-induced EMT with the upregulation of Snail, vimentin, N-cadherin, and downregulation of E-cadherin, which might play a vital role in maintaining the mitochondrial function of intrahepatic cholangiocarcinoma (ICC) [32]. In our study, DDP treatment induced p62 expression was suppressed by drug combination treatment, suggested that tumor tissue cells may be inhibited gradually developing DDP resistance and EMT. In subsequent research, we may conduct an animal model of ascites metastasis of OC to focus on cancer metastasis.

Autophagy is an intracellular metabolic process, which may be caused by oxidative stress plays an important role in the development of tumors, participates in the energy metabolism and material metabolism of tumor cells, changes MMP and related metabolites including ATP, ROS, SOD and so on, as well as affect the changes of tumor growth microenvironment [33, 34]. First of all, mitochondria are the energy center and regulator of cells. The decline in MMP is a landmark event in the early stages of cell apoptosis. In our study, SB-HD increased the proportion of early and late apoptosis in SKOV3 and OVCAR3 cells, and decreased MMP compared with Control. Compared with DDP alone, the combination treatment significantly increased the proportion of apoptosis and further promoted the decrease of MMP. In addition, ATP is an intracellular energy molecule and an important regulator of autophagy, and it can also regulate the immune response in the tumor microenvironment, enhance the body’s immune surveillance and elimination of tumors [35]. So, we examined the changes of ATP in SB-HD combined with DDP subsequently, and found that combination treatment could reduce ATP levels in SKOV3 and OVCAR3 cells. It is known that the levels of ATP are closely related to the degree of autophagy in tumor cells. When tumor cells are in a low energy state, the level of ATP decreases, which can activate AMPK signaling pathway and promote the initiation of autophagy [36]. As expected, our Western blotting results showed autophagy-associated proteins to be induced in the combination treatment.

Several studies have reported that mitochondria are the main sites of oxygen free radicals in cells, and also an important target of oxidative stress. Excessive levels of oxygen free radicals can lead to mitochondrial dysfunction and DNA oxidative damage. ROS can play a dual role in anti-tumor by affecting mitochondrial function, the effect depends on the levels of ROS and the state of the cells [37, 38]. In this study, we found that ROS content in cells treated with SB-HD extract alone and combination treatment showed a significant downward trend, and there was no significant difference between SB-HD extract alone and combination treatment. According to the existing research, ROS plays an important role in promoting cell mitosis, which is indispensable to induce cell proliferation. When ROS accumulates in cells, it can induce apoptosis due to oxidative stress, but it is less toxic to the corresponding normal cells. When ROS is at a low level, the cells will be in a state of proliferation inhibition, but the corresponding normal cells are less sensitive to it. The difference of drug sensitivity between tumor cells and normal cells may be due to the high levels of ROS expression in tumor cells themselves. Our results suggest that combination treatment reduce the levels of ROS in OC cells, which lead to cell cycle inhibition.

From a biological function, SOD is a kind of metalloproteinase in mitochondria, which is a powerful oxygen free radical scavenger. Its main function is to remove superoxide anion (O2-) in cells and prevent the occurrence of oxidative damage. It has been reported that SB has immunomodulatory effect and anti-lipid peroxidation effect, which can scavenge oxygen negative free radicals and improve the activity of SOD, and its antioxidant effect has been widely accepted. HD has also been found to increase the activity of SOD in gastric tissue, reduce the content of MDA, and have a protective effect on gastric ulcer and other gastrointestinal membrane damage. Therefore, we speculated that SB-HD assisted DDP to down-regulate ROS in OC, SOD activity may play an important role. Subsequently, we detected the SOD activity in each group of cells treated with different drugs, and found that combination treatment increased the activity of SOD, which proved our inference. In tumor cells, SOD activity is usually low, and studies have shown that the occurrence of some cancers is related to the decline of SOD levels in vivo. By increasing the levels of SOD, it is expected to provide new ideas for cancer prevention and treatment.

Ferroptosis is a new way of cell death, which is different from the traditional ways of cell death such as necrosis and apoptosis, and was first proposed in 2012 [39]. Recent studies have pointed out that ferroptosis may be autophagy-dependent, because the response to ferroptosis activators can lead to the accumulation of autophagosomes, and the components of autophagy mechanism contribute to the occurrence of ferroptosis [14]. In addition, FTH1 encodes a ferritin heavy chain, the autophagic degradation of FTH1 can increase oxidative stress and the level of Fe3+ in cells, making cells more sensitive to ferroptosis [40]. At the same time, ferroptosis depends on the accumulation of intracellular iron and ROS [41, 42]. Iron-dependent lipid peroxidation is the main feature of cell ferroptosis, and the damage of cell membrane caused by lipid peroxides is an important reason for ferroptosis [41, 42]. Besides, nuclear receptor coactivator 4 (NCOA4) is a selective receptor that maintains iron homeostasis by binding to ferritin, transporting it to the lysosome, and promoting autophagy degradation, and is considered as an important regulator of ferroptosis [43, 44]. It has been reported that a decrease in intracellular Fe2+/oxidative stress response and an increase in glutathione level are accompanied by an decrease in NCOA4 expression, revealed that the level of oxidative stress is closely related to NCOA4 [44]. In our study, we have preliminarily found an increase in this protein expression by combination treatment, this may also imply an increase in oxidative stress. According to the results, combination treatment reduces the level of intracellular ROS, we speculate that although SB-HD can enhance the sensitivity of DDP chemotherapy in OC by regulating oxidative stress and activating autophagy, but it is may not further promote the occurrence of cell ferroptosis. Because the high activity of SOD enzyme generally indicates that there are too many free radicals in the body. ROS is a series of active oxygen free radicals produced by cells in the process of metabolism, which is essentially a chemical substance with unpaired electrons and has a strong ability to compete for electrons. A large amount of ROS will lead to the decrease of MMP and ATP content, then promote cell apoptosis and the destruction of mitochondrial homeostasis. Mitochondria are important mediators of cellular metabolism, producers and targets of ROS. The disruption of mitochondrial homeostasis leads to the decrease of ROS follow-up production, and along with the increase of SOD activity further reduces ROS levels, meanwhile, autophagy also acts as a ROS scavenger in cancer cells. To sum up, the dynamic changes mentioned above may regulate OC cells in a state of oxidative stress, which is a joint effect by regulating autophagy, affecting MMP, ROS production, ATP content and SOD activity.

NRF2/HO-1 pathway plays an important role in the occurrence and regulation of oxidative stress and inflammatory response [45,46,47]. Some studies suggest that NRF2/HO-1 is involved in regulating autophagy and cell apoptosis [48,49,50,51]. In the preliminary research of our team, NRF2 and HO-1 were significantly increased in the OC cells treated with SB-HD alone by proteome and transcriptome analysis, simultaneously were validated by Western blotting and quantitative PCR (qPCR) experiments with or without inhibitors Fer-1, ZnPP and 3-MA. Research has illustrated that ferritinophagy may be the key reason SB-HD induces ferroptosis in OC cells and thus exert anti-tumor effects. In this study, we first found NRF2 and HO-1 in the tumor tissue was significantly increased by IHC stained, and also detected the combination treatment significantly increased the expression of NRF2 and HO-1 both in OC cells and tumor tissue of mice by Western blotting experiments. Moreover, we found that combination treatment reduced the expression of FTH1 in OC cells and tumor tissues through autophagy. Next, to further clarify the molecular mechanism, three inhibitors of Fer-1, ZnPP and 3-MA were used to extract cellular proteins on the basis of the combination treatment in this part of the experiment. 3-MA can effectively reverse the expression changes of the target proteins. Fer-1 can obviously reverse the expression of NRF2, HO-1, and FTH1 in this signaling pathway. Therefore, we conclude that the mechanism by which SB-HD increases sensitivity to DDP chemotherapy in OC may be primarily associated with ferroptosis related oxidative stress and autophagic degradation of ferritin. Interestingly, we also found that HO-1 decreased in SKOV3 cells after the use of the inhibitor Fer-1, and increased in OVCAR3 cells after the use of the inhibitor ZnPP. As Fer-1 is a synthetic antioxidant, it mainly prevents the damage of membrane lipids through a reductive mechanism, thereby inhibiting cell death. Therefore, we speculated that the addition of Fer-1 to SKOV3 cells may compensate for part of the antioxidant activity of the cells originally produced by HO-1, which result in decreasing HO-1 expression. However, ZnPP is a competitive inhibitor of HO-1 which has anticancer activity. The concept of competitive inhibitor refers to the inhibitor that produces competitive inhibition, which usually has structural similarity with the substrate of the inhibited enzyme, and can compete with the substrate for the binding site on the enzyme molecule, thus producing reversible inhibition of enzyme activity. In this study, the inhibitor ZnPP was added to inactivate HO-1, which may destroy the important physiological and pathological functions of HO-1 involved in regulation, and then trigger more HO-1 expression in positive feedback, so the detected HO-1 expression level increased significantly.

Finally, due to the complex clinical features of OC, we also analyzed the key target genes by bioinformatics utilizing online large databases, hoping to return the problems in clinical practice to the clinical cases for further analysis and exploration, in order to clarify the correlation between target genes and OC, the expression differences between healthy people and patients, the expression level in clinical stages and the prognosis. We found that the abnormal expression levels of NRF2 and FTH1 mRNA showed high prognostic value. The other four target genes of this signaling pathway HO-1, p62, ATG5 and LC3, also play an important role in the development of OC, either individually or by interacting with NRF2 and FTH1. We conclude that NRF2 and FTH1 may become potential biomarkers for individualized prediction and treatment of OC. It may provide a reference for the potential study of DDP combined with other herb drugs to enhance the chemosensitivity of OC.