Study On The Anti-inflammatory Effect Of Maslinic Acid On IPEC-J2 Cell And Antitumor Mechanism On HCT-116 And HepG2 Cells

Maslinic acid(MA)is a natural pentacyclic triterpene,and it has anti-inflammatory,antitumor,cardioprotective,neuroprotective,antioxidant and antiviral effects.Interestingly,MA has different effects on the same signaling pathway under different conditions,thus playing a protective role.The purpose of this study was to explore the common mechanism of MA playing different biological activities in the LPS inflammation model of normal intestinal cell line IPEC-J2,colorectal cancer cell line HCT-116 and hepatocllular carcinoma cell line HepG2.First,this study selected normal intestinal cell line to explore the inflammatory protective effect of MA.1.The cell viability measured by CCK-8 was used as the basis for concentration selection.After 24 h of MA treatment,low concentrations promoted cell viability,while high concentrations showed an inhibitory effect,and the concentration of 0.5 μg/m L,which exhibited the best promoting effect(P < 0.05),was chosen for subsequent experiments.LPS,at a concentration of 1 μg/m L forming the inflammation model,and the anti-inflammatory effects of MA were investigated by cotreatment with these two substances.2.The differential expression genes were classified by transcriptome sequencing.One of the clusters shown LPS downregulated gene expressions and effectively recovered by MA,the highest enrichment was focal adhesion signaling pathway through KEGG signaling pathway enrichment.Furthermore,the changes of gene expression in focal adhesion signaling pathway was scrutinized via PCR array.Compared with the control group,there was a significant decrease in gene expressions induced by LPS.However,MA could alleviate the decrease of gene expression and restoring it to levels similar to that observed with MA treatment alone,that also consistent with the trend of sequencing results.The genes determined were labeled on signaling pathway map,revealing FAK as the key effector protein in this pathway.3.Western blotting results showed that LPS significantly inhibited the expression of FAK protein(P < 0.05),while MA maintained the stability of the pathway by promoting FAK protein phosphorylation(P < 0.05).Furthermore,the ability of MA to reverse LPS-induced cell migration was confirmed through cell scratch test and immunofluorescence of Vinculin and F-actin.4.To further confirm that MA exerts a protective role by promoting FAK phosphorylation,a FAK phosphorylation inhibitor,Defactinib,was introduced to verify the aforementioned experiment results,ultimately determining that FAK phosphorylation constitutes the main protective effect of MA.5.To elucidate the regulatory mechanism of MA on FAK protein,potential MA targets were identified through molecular docking,uncovering its potential to bind to PTEN and PKC proteins,which regulate FAK phosphorylation.Co-immunoprecipitation and western blotting were used to analyze the protein binding to FAK.The results showed that MA may bind to PTEN protein and significantly inhibit its interaction with FAK and promote FAK phosphorylation(P < 0.05).Meanwhile,the results of molecular docking showed that MA could also bind to PKC protein,but the binding effect was not found in low concentration treatment.Inflammatory lesions of the intestine can lead to colorectal cancer,and MA not only has anti-inflammatory effect,but also has excellent antitumor effect.In the second part,we selected the colorectal cancer cell line HCT-116 and hepatocellular carcinoma cell line HepG2,representing a common site of colorectal cancer metastasis,to investigate whether MA exerts its antitumor effects by regulating the phosphorylation of FAK protein.1.Cell viability measured by CCK-8 and crystal violet cell counting used as the basis for concentration selection,0.5 μg/m L MA had no significant effect on the viability and number of the two cell lines,but 10 μg/m L MA began to significantly inhibit these.The half-maximal inhibitory(IC50)concentration of MA in HCT-116 cells was determined to be 36 μg/m L,while in HepG2 cells,it was 35 μg/m L.The IC50 concentration of the two cell lines could significantly inhibit DNA replication by Ed U staining(P < 0.05),and this concentration was selected for proteomics.2.The outcomes from the proteomics downregulation and the enrichment of tumor-related differential expression proteins revealed that focal adhesion signaling pathway was the most highly enriched pathway in both two cell lines.Additionally,the IC50 concentration of MA led to downregulation of the gene transcription in the focal adhesion signaling pathway.The results of molecular docking showed that the regulatory factors of FAK phosphorylation,PTEN and PKC,were candidate targets of MA.3.Subsequent co-immunoprecipitation and western blotting assays demonstrated that at a concentration of 0.5 μg/m L,MA significantly inhibited the interaction between FAK and PTEN(P < 0.05).There was no significant difference in the interaction between FAK and PKC protein,and there was no significant change in the expression and phosphorylation of FAK and its downstream proteins.With the increase of 10μg/m L concentration of MA,it significantly inhibited the interaction between FAK and PKC protein(P < 0.05),blocked the phosphorylation of FAK(P < 0.05),and inhibited the phosphorylation of m TOR,AKT,JNK,ERK,GSK3 B and the protein expression of PI3 K,CTNNB1,CCND1,BCL2 and CDC42.The effect of MA at IC50 concentration was more significant.4.The ability of cell migration was evaluated by cell scratch test and immunofluorescence of Vinculin and F-actin.It was found that there was no significant difference between 0.5 μg/m L MA group and control group.In contrast,the10 μg/m L and IC50 concentration of MA not only weakened the migration ability of cancer cells but also induced cell detachment and cell death(P < 0.05).5.The effects of MA on cancer cell survival and proliferation were evaluated by flow cytometry.At0.5 μg/m L,there were no significant differences compared to the control group,but at10 μg/m L and the IC50 concentration of MA,cell cycle arrest was observed,cancer cells significantly stagnated in G1 phase(P < 0.01)and S phase cells decreased significantly(P < 0.05).Moreover,the IC50 concentration of MA also significantly increased the number of apoptotic cells(P < 0.05).The apoptosis of adherent cancer cells in suspension culture also increased with the increase of MA concentration,but the difference was gradually reduced compared with normal adherent cells.At the IC50 concentration of MA,there was no significant difference between the two groups.In addition,IC50 concentration of MA significantly promoted the interaction between FAK and Notch1 protein(P < 0.05),inhibited the expression of HES1 and HES5 protein,further confirming the occurrence of anoikis.Notably,the results of molecular docking demonstrated that MA shared a similar binding site with the PKC protein inhibitor 3ZK.It was found that the antitumor effects of MA on colorectal cancer and hepatocellular carcinoma cells mainly through the inhibition of the PKC protein.Thus,MA can be considered a potential PKC protein inhibitor in the cancer therapy.In this study,we determined the common regulatory mechanism of antiinflammatory and antitumor effects of MA,that is may through binding with PTEN and PKC proteins,regulating FAK protein phosphorylation,affecting the expression of genes and proteins in focal adhesion signaling pathway.These regulatory effects consequently impact cell migration,proliferation,and survival.Notably,at low concentrations,MA may primarily bind to the PTEN protein,and it might begin to bind to PKC protein with the increase of concentration,showing a completely different effect.In normal intestinal cells,low concentration of MA promotes FAK phosphorylation and maintains the homeostasis of focal adhesion signaling pathway.In colorectal cancer and hepatocellular carcinoma cells,high concentration MA inhibited FAK phosphorylation and showed similar tumor inhibitory effect.This study found a common target for the biological action mechanism of MA,explained the reasons for the opposite effect of MA through the action target at different concentrations,predicted the possible action mode of this regulation mechanism,and hoped to offer insights into the exploration of the action mechanism of bioactive components.