Qingjie Fuzheng granules inhibit colorectal cancer cell growth by the PI3K/AKT and ERK pathways

BACKGROUND Qingjie Fuzheng granules(QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, including colorectal cancer(CRC).However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs' anticancer effect have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of colorectal cancer cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways.AIM To better evaluate the mechanism underlying the anti-cancer effect of QFGs on the CRC cell lines HCT-116 and HCT-8.METHOD First, we measured cell viability and cytotoxicity by performing MTT and lactate dehydrogenase(LDH) assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation and analyzing Hoechst 33258 staining. Second, cell cycle and apoptosis rates were measured by fluorescence activated cell sorting, and the expression levels of survivin, cyclin D1, CDK4, p21,Bax, Bcl-2, Fas, FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blots and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were used to elucidate the specific apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3 K/AKT and ERK signaling pathwayswas examined using the western blot assay to investigate the possible mechanism.RESULTS MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by(6.90% ± 1.03%)–(59.70% ± 1.51%)(HCT-116; P < 0.05)and(5.56% ± 4.52%)–(49.44% ± 2.47%)(HCT-8; P < 0.05), and cytotoxicity was increased from 0.52 ± 0.023 to 0.77 ± 0.002(HCT-116; P < 0.01) and from 0.56 ±0.054 to 0.81 ± 0.044(HCT-8; P < 0.01) compared with the non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells.QFGs also increased the expression levels of Bax, Fas and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast, when adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3 K, AKT and ERK.CONCLUSION These results demonstrated that QFGs can inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3 K/AKT and ERK signaling pathways.

Bioinformatic Analysis and Experimental Verification of QJHGD on Caerulein-induced Inflammatory Response in SAP Model Rats Based on TLR4/NF-κB/My D88 Pathway

[Objectives]To conduct bioinformatic analysis and experimental verification of Qingjie Huagong Decoction(QJHGD)on caerulein-induced inflammatory response in severe acute pancreatitis(SAP)model rats based on TLR4/NF-κB/MyD88 pathway.[Methods]The effective component groups and potential targets of QJHGD were collected by the network pharmacology method.A drug-component-target network was constructed.The GO and KEGG of targets were enriched and analyzed with the aid of Metascape database,and the target pathway related to SAP inflammation was screened.The SAP rat model was established by caerulein combined with lipopolysaccharide,and QJHGD was intragastrically administered.Pancreatic tissue was observed by HE staining.In addition,enzyme-linked immunosorbent assay and immunohistochemistry were used to verify the anti-inflammatory effect of QJHGD on SAP rats and its regulatory effect on TLR4/NF-κB/MyD88 target pathway.[Results]A total of 105 active components of QJHGD and 148 key targets of SAP were predicted and screened;KEGG was enriched in 320 different pathways including toll-like receptor and NF-κB classical pathways.Animal experiment verified that QJHGD reduced serum amylase,serum lipase activity,IL-6,TNF-αlevels in SAP rats;HE staining showed the effect of QJHGD on the pathological changes of pancreas,and QJHGD inhibited the positive expression of key proteins of TLR4,NF-κB and MyD88 in the inflammatory transduction pathway.[Conclusions]The mechanism of QJHGD improving pancreatic injury in SAP rats may be related to down-regulating the expression of key proteins in the TLR4/NF-κB/MyD88 pathway.