| BackgroundEsophageal adenocarcinoma,a highly malignant upper gastrointestinal cancer,ranks among the most prevalent histological subtypes of esophageal cancer.Its incidence in China has been steadily increasing,currently approximating 0.5-1/100,000.Despite advancements in surgical,radiotherapeutic,and chemotherapeutic surgeries,the five-year survival rate for most patients with esophageal adenocarcinoma remains below 20%.The multifaceted pathogenesis of this disease,involving factors such as gastroesophageal reflux and abdominal obesity,remains incompletely understood,necessitating the identification of therapeutic targets and novel treatment agents.In contrast to ancient Chinese medicine literature that briefly mentions the characteristics of esophageal cancer,such as"Suwen.Zhizhenyaodalun," which notes:"If the ingested diet encounters obstruction within the esophagus,it shall precipitate emesis," limited information exists on the specific pathogenesis and progression of esophageal adenocarcinoma,leaving the traditional Chinese medicine(TCM)basis for this cancer subtype inadequately explored.However,a research group led by a professor at a prominent hospital,drawing upon extensive clinical experience,has consistently observed that esophageal adenocarcinoma often manifests the TCM syndrome of phlegm-heat.As a result,we proposed the "phlegm-heat binding chest"theory as an essential therapeutic approach for esophageal adenocarcinoma.In clinical practice,the modified Xiaoxianxiong Decoction(MXD),a formulation comprising four TCM herbs,namely Gualou,Coptidis Rhizoma,Pinellia,and Turmeric,has exhibited promising outcomes,ameliorating symptoms and prolonging survival.The function of MXD is believed to be related to clearing heat and dissolve phlegm,moving qi and invigorate blood,and loosening the chest and dessepate binds.Modern investigations have unveiled a molecular basis for the phlegmheat syndrome,implicating blood lipid metabolism disorders,with Xiaoxianxiong Decoction as a potent regulator of blood lipid metabolism.Furthermore,the blood lipid metabolism disorder serves as an important factor that affects the lipid metabolism of tumor tissue and affects its occurrence and development.Notably,preliminary experiments indicated that MXD exerted minimal direct anti-tumor effects on esophageal adenocarcinoma developed in normal diet mice,in contrast to the standard Xiaoxianxiong Decoction,which has been reported to possess such effects.Consequently,it was surmised that MXD’s impact on esophageal adenocarcinoma might not stem from direct tumor suppression but rather from the correction of the influence of dyslipidemia to tumor itself.On this basis,our study aimed to explore the effect and underlying mechanisms of MXD on high-fat-induced esophageal adenocarcinoma in vivo and in vitro,with a particular emphasis on dyslipidemia.This comprehensive investigation encompassed clinical research in conjunction with in vivo and in vitro experiments,with the ultimate objective of illuminating the potential therapeutic utility of MXD in esophageal adenocarcinoma treatment.Objectives1.This study aimed to comprehensively elucidate the symptoms and characteristics presented by patients diagnosed with esophageal adenocarcinoma.By analyzing the prevalence of dyslipidemia and syndrome elements related to phlegm-heat,we sought to establish a correlation between these factors.The ultimate goal was to provide essential data references for the treatment of esophageal adenocarcinoma using an integrated approach that combined TCM and Western medicine.2.Through the induction of a blood lipid metabolism disorder model via high-fat exposure in vivo and in vitro,this phase of the study aimed to explore the impact of this model on the lipid metabolism of esophageal adenocarcinoma and the initiation and progression of it.3.In this stage,the focus lay in intervening in the esophageal adenocarcinoma model,characterized by lipid metabolism disorder induced by high-fat exposure both in vivo and in vitro.The primary objective was to elucidate the underlying mechanisms by which MXD exerts its therapeutic effects on esophageal adenocarcinoma by regulating lipid metabolism,in order to establish a scientifically sound basis for the clinical application of MXD.Methods1.Correlation between TCM syndrome elements and dyslipidemia in patients with esophageal adenocarcinomaThis study enrolled patients diagnosed with esophageal adenocarcinoma at a hospital between January 2010 and March 2023.Comprehensive patient data,including basic information,blood lipid profiles,and TCM syndrome elements,were retrospectively collected.The prevalence of syndrome elements and dyslipidemia in the patient cohort was calculated,and their relationship was analyzed.2.In vivo experiment:Effect of MXD on esophageal adenocarcinoma2.1 Inhibitory effect of MXD on high-fat diet-induced OE33 cell xenograft tumorTwenty-four NOD/SCID mice were randomly divided into four groups:the high-fat control group,high-fat low-concentration MXD group,high-fat high-concentration MXD group,and control group.The former three groups were fed a high-fat diet,while the control group received a standard diet.After eight weeks of dietary intervention,OE33 cells were employed to induce tumor formation in the mice.Subsequently,the mice were intragastrically administered low-concentration MXD(high-fat low-concentration MXD group),highconcentration MXD(high-fat high-concentration MXD group),or distilled water(high-fat control group and control group)on the 3rd d after tumor induction for a five-week observation period.Throughout this period,the body weight,body length,Lee’s index,abdominal circumference,subcutaneous tumor volume,tumor weight and serum TG level of mice in each group were observed and recorded.2.2 Effect of MXD on lipid metabolism and the PI3K/AKT/mTOR pathway in esophageal adenocarcinoma xenografts in mice induced by a high-fat dietLipidomics analysis was performed on tumor tissues obtained from each group of mice.Moreover,Western blotting analysis was conducted to detect the expression levels of key lipid metabolism proteins(CD36,ACLY,SREBP1,and FASN)and proteins within the phosphatidylinositol 3-kinase/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway in the tumor tissues of mice.Through these analyses,the effects of the high-fat diet and the application of MXD on lipid profiles and the expressions of key proteins involved in lipid metabolism and proteins within the PI3K/AKT/mTOR pathway were investigated.3.In vitro experiment:Effect of MXD on esophageal adenocarcinoma3.1 Screening procedure for establishing the OE33 cell model in a high-fat environmentOE33 cells were exposed to different concentrations(0,0.5 μM,5 μM,50 μM,100 μM,200 μM,400μM,and 800 μM)of oleic acid,palmitic acid,and a 2:1 mixture of oleic acid and palmitic acid 24 h,48 h,and 72 h.Cell viability was evaluated using the Cell Counting Kit-8(CCK-8)method,triglyceride(TG)content in OE33 cells was assayed using a TG kit,and the formation of lipid droplets in OE33 cells was analyzed through oil red O staining.Based on the above results,the optimal conditions for establishing the OE33 cell model in a high-fat environment were determined.3.2 Effect of MXD-containing serum on the activity of high-fat diet-treated OE33 cellsDifferent concentrations of MXD-containing serum and blank serum was prepared by gavage rats with different doses of MXD or distilled water;OE33 cells were divided into four groups:the high-fat blank serum group,high-fat low-concentration MXD group,high-fat highconcentration MXD group,and blank serum group.After a 48h exposure to a high-fat or normal environment,cells in each group were further treated with a complete medium containing 10%different concentrations of the MXD-containing serum or blank serum for 48h.Cell proliferation activity in 24h and 48h was assessed using the CCK-8 method,and cell colony formation in each group was observed by plate cloning experiments.The OE33 cells were then divided into a traditional Chinese medicine intervention group and a blank serum group.After treated in a high-fat environment for 48 hours,cells were intervened with the complete medium prepared by high-dose MXD containing serum or blank rat serum,respectively.The cell apoptosis and cell cycle of the two groups were detected by flow cytometry at 48h.3.3 Effects of MXD-containing serum on lipid metabolism and the PI3K/AKT/mTOR pathway in high-fat treated OE33 cellsWestern blotting analysis was performed to detect the expressions of key proteins related to lipid metabolism(CD36,ACLY,SREBP1,and FASN)and those related to the PI3K/AKT/mTOR pathway in the high-fat blank serum group,high-fat low-concentration MXD group,high-fat high-concentration MXD group,and blank serum group.The content of TGs in each group of cells was measured using a TG kit,and lipid droplet formation in each group of cells was observed through oil red O staining.3.4 Effect and mechanism of MXD-containing serum on PI3K/AKT/mTOR pathway in FASN overexpressed OE33 cells treated with high-fat environmentFASN overexpression(FASN OE)OE33 cells and empty vector(Vector)OE33 cells were constructed,and the proliferation activity of FASN OE and Vector cells in high-fat environment was detected by CCK-8 method at 24h,48h and 72h respectively;The effect of FASN inhibitors C75 and cerulenin on the viability of OE33 cells in high lipid environment under the condition of 24h and 48h was also detected.The protein expression of PI3K/AKT/mTOR pathway in FASN OE and Vector cells was detected by Western blot,and the effects of FASN inhibitors C75 and cerulenin on the expression of PI3K/AKT/mTOR pathway protein in OE33 cells in high-fat environment were detected.The cells were further divided into blank serum+Vector group,MXD-containing serum+Vector group and MXD-containing serum+FASN OE group.Western blot was used to detect the expression of FASN and PI3K/AKT/mTOR pathway proteins in each group.Results1.The present study examined a cohort of 45 patients diagnosed with esophageal adenocarcinoma,revealing a notable dyslipidemia rate of 77.78%among them.Among the subset of 33 patients with available case data,66.67%were diagnosed with phlegm-heat syndrome based on syndrome elements.Remarkably,all patients diagnosed with esophageal adenocarcinoma and phlegm-heat syndrome demonstrated a considerably high frequency of dyslipidemia,with a prevalence as high as 90.91%,characterized primarily by elevated TG content.Logistic regression analysis identified elevated TG content as a prominent risk factor for phlegm-heat syndrome(OR=12.0,95%CI 1.30-110.53).2.Following an eight-week dietary intervention,mice exposed to a high-fat diet(n=18)exhibited significantly elevated body weight,Lee’s index,and abdominal circumference compared with mice exposed to a standard diet(n=6)(P<0.05).However,no significant difference in body length was observed between the two groups(P>0.05).After tumor induction,the high-fat low-concentration MXD group and the high-fat high-concentration MXD group displayed reduced body weight in contrast to the high-fat control group(P<0.05).Notably,the growth of tumor volume in all three high-fat diet groups was markedly accelerated compared with that in the control group.Nevertheless,as the dose of MXD increased,the growth of tumor volume in three high-fat diet groups decelerated,with the high-fat highconcentration MXD group demonstrating significant tumor group inhibition.At the end of the observation period,the order of tumor volume,from largest to smallest,was as follows:the high-fat diet control group,the high-fat diet low-concentration MXD group,the control group,and the high-fat diet high-concentration MXD group.The serum TG levels of the mice in each group was compared in this study.The results showed that the high-fat diet control group had a significantly higher TG level than the control diet group(P<0.01),meanwhile,the highconcentration MXD could significantly reduce the serum TG level(P<0.01).The lipidomics analysis unveiled a significant increase in the relative content of TGs and free fatty acids in the tumor tissue of mice subjected to a high-fat diet(P<0.05).However,treatment with MXD effectively counteracted this effect,yielding a substantial reduction in TG and free fatty acid levels(P<0.05).Additionally,MXD restored the perturbed levels of phosphatidylcholine and phosphatidylethanolamine in tumor tissue induced by the high-fat diet.Western blotting analysis revealed a remarkable upregulation in the expressions of pivotal proteins involved in lipid metabolism,namely CD36,SREBP1,and FASN,as a consequence of the high-fat diet(P<0.05).Concomitantly,the high-fat diet significantly promoted the expressions of proteins within the PI3K/AKT/mTOR pathway,including PI3K,phosphorylated(p)-AKT,and p-mTOR(P<0.05).In contrast,intervention with MXD downregulated the expression of FASN with statistical significance(P<0.05)but had no statistically significant effect on the expressions of CD36,ACLY,and SREBP1(P>0.05).Moreover,MXD could significantly inhibit the expressions of PI3K,p-AKT,and p-mTOR(P<0.05).3.Among the diverse concentrations examined,the intervention using a mixture of 50 μM oleic acid+25 μM palmitic acid for 48 h exhibited a significant promotion of OE33 cell activity,leading to a noteworthy increase in lipid droplet formation(P<0.05)and elevated cholesterol levels(P<0.05)within the cells.Thus,this intervention was selected as the optimal method to establish the high-fat diet-induced OE33 cell model.At the 48-h time point,MXD-containing serum demonstrated a remarkable inhibitory effect on OE33 cells in the high-fat environment(P<0.05),with this inhibitory effect showing a dose-dependent trend.However,at the 24-h time point,the inhibitory effect was less pronounced(P>0.05).Furthermore,the MXDcontaining serum significantly suppressed the formation of OE33 cell colonies in a dosedependent manner(P<0.05).Evaluation of cell apoptosis and cell cycle via flow cytometry unveiled that the MXD-containing serum significantly promoted the apoptosis of OE33 cells in the high-fat environment(P<0.05)and induced OE33 cell cycle arrest in the G0/G1 phase(P<0.05).Investigation of the expressions of key lipid metabolism proteins and PI3K/AKT/mTOR pathway proteins in different groups revealed that the high-fat environment significantly upregulated the expressions of CD36 and FASN in OE33 cells(P<0.05),but it had no significant effects on ACLY and SREBP1(P>0.05).Conversely,the MXD-containing serum significantly inhibited the expression of FASN(P<0.05)while exhibiting no significant differences in effects on CD36,ACLY,and SREBP1(P>0.05).Additionally,the MXDcontaining serum notably suppressed the expressions of PI3K,p-AKT,and p-mTOR(P<0.05),especially at high doses.By comparing the proliferation activity of FASN OE and Vector cells at 24h,48h,and 72h in a high-fat environment,it was found that the proliferation activity of FASN OE cells at 72h was significantly enhanced compared with Vector cells(P<0.05);at the same time,FASN inhibitors C75 and cerulenin can significantly inhibit the activity of OE33 cells in a high-fat environment in a concentration-dependent manner(P<0.01).In addition,the p-AKT and pmTOR protein levels in FASN OE cells were significantly up-regulated compared with Vector cells in high-fat environment(P<0.01),while FASN inhibitors C75 and cerulenin could significantly down-regulate the proteins levels of FASN,p-AKT and p-mTOR.Rescue experiment was then performed in this study,the results showed that the overexpression of FASN could not only weaken the inhibitory effect of the MXD-containing serum on the activity of OE33 cells,but also weaken its inhibition effect on the protein levels of FASN,p-AKT and p-mTOR.Conclusion1.Patients diagnosed with esophageal adenocarcinoma and presenting with phlegm-heat syndrome exhibit a significant association with lipid metabolism disorder.Elevated TG levels,a prominent feature of lipid metabolism disorder,may serve as the underlying basis for phlegmheat syndrome in these patients.2.The blood lipid metabolism disorder characterized by elevated TG,the lipid metabolism disorder in tumor tissue mainly manifested by the up-regulation of CD36 and FASN protein,accompanied by the accumulation of lipid and the reorganization of lipid profile,as well as the activation of PI3K/AKT/mTOR pathway may be involved in the promotion of esophageal adenocarcinoma growth.3.In this context,MXD emerges as a potential therapeutic agent capable of inhibiting esophageal adenocarcinoma.It exerts effects by reducing the lipids in blood and lipid accumulation in tumor cells,as well as by downregulating the expression of FASN,which leads to a subsequent inhibition of the PI3K/AKT/mTOR pathway. |
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