Ganoderic acids alleviate atherosclerosis by inhibiting macrophage M1 polarization via TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND AND AIMS: Atherosclerosis (AS) is a chronic inflammatory disease characterized by lipid infiltration and plaque formation in blood vessel walls. Ganoderic acids (GA), a class of major bioactive compounds isolated from the Chinese traditional medicine Ganoderma lucidum, have multiple pharmacological activities. This study aimed to determine the anti-atherosclerotic effect of GA and reveal the pharmacological mechanism. METHODS: ApoE-/- mice were fed a high-cholesterol diet and treated with GA for 16 weeks to induce AS and identify the effect of GA. Network pharmacological analysis was performed to predict the anti-atherosclerotic mechanisms. An invitro cell model was used to explore the effect of GA on macrophage polarization and the possible mechanism involved in bone marrow dereived macrophages (BMDMs) and RAW264.7 cells stimulated with lipopolysaccharide or oxidized low-density lipoprotein. RESULTS: It was found that GA at 5 and 25 mg/kg/d significantly inhibited the development of AS and increased plaque stability, as evidenced by decreased plaque in the aorta, reduced necrotic core size and increased collagen/lipid ratio in lesions. GA reduced the proportion of M1 macrophages in plaques, but had no effect on M2 macrophages. In vitro experiments showed that GA (1, 5, 25 µg/mL) significantly decreased the proportion of CD86+ macrophages and the mRNA levels of IL-6, IL-1ß, and MCP-1 in macrophages. Experimental results showed that GA inhibited M1 macrophage polarization by regulating TLR4/MyD88/NF-κB signaling pathway. CONCLUSIONS: This study demonstrated that GA play an important role in plaque stability and macrophage polarization. GA exert the anti-atherosclerotic effect partly by regulating TLR4/MyD88/NF-κB signaling pathways to inhibit M1 polarization of macrophages. Our study provides theoretical basis and experimental data for the pharmacological activity and mechanisms of GA against AS.

[Ginsenoside Rh2 induces apoptosis and autophagy of K562 cells by activating p38].

To study the effect of ginseng saponin Rh2 in inducing apoptosis of human leukemia K562 cells, and explore its mechanism from the aspect of autophagy pathway. CCK-8 assay was used to examine the growth inhibition of human leukemia cell lines K562 treated with ginsenoside Rh2; flow cytometry (FCM) was used to detect cell apoptosis; Hoechst staining was used to observe the changes of cell morphological apoptosis; Acridine and MDC staining were used to detect the effects of the Rh2 on autophagy; Western blot and RT-PCR were used to detect the expression levels of the proteins closely associated with autophagy and apoptosis. In order to study the effect of autophagy in proliferation and apoptosis, we used the autophagy inhibitor (3-MA).CCK-8 indicated that Rh2 at low concentration could effectively inhibit the proliferation of leukemia cellsin dose- and time-dependent manners in K562 cells; FCM indicated that Rh2 induced apoptosis; Hoechest staining showed that K562 cells had typical apoptotic morphological changes by treated Rh2; Acridine and MDC staining showed that Rh2 enhanced the green fluorescence and a large number of acidic autophagy vesicles were present; Western blot and RT-PCR results showed that Rh2 increased the expression levels of Beclin-1, LC3A, LC3B, activated Caspase-3 and p-p38 in K562 cells; application of autophagy inhibitors(3-MA) could weaken the inhibition effect of Rh2 on proliferation and induction effect on apoptosis in K562 cells. Ginsenoside Rh2 inhibited the proliferation and induced apoptosis probably through activating p-p38, and inducing cell autophagy signaling pathway in K562 cells.

20(S)-ginsenoside Rh2 inhibits the proliferation and induces the apoptosis of KG-1a cells through the Wnt/ß-catenin signaling pathway.

Previous research has shown that total saponins of Panax ginseng (TSPG) and other ginsenoside monomers inhibit the proliferation of leukemia cells. However, the effect has not been compared among them. Cell viability was determined by Cell Counting Kit-8 assay, and ultra-structural characteristics were observed under transmission electron microscopy. Cell cycle distribution and apoptosis were determined by flow cytometry (FCM). Real-time fluorescence quantitative­PCR, western blotting and immunofluorescence were used to measure the expression of ß-catenin, TCF4, cyclin D1 and NF-κBp65. ß-catenin/TCF4 target gene transcription were observed by ChIP-PCR assay. We found that 20(S)-ginsenoside Rh2 [(S)Rh2] inhibited the proliferation of KG-1a cells more efficiently than the other monomers. Moreover, (S)Rh2 arrested KG-1a cells in the G0/G1 phase and induced apoptosis. In addition, the levels of ß-catenin, TCF4, cyclin D1 mRNA and protein were decreased. The ChIP-PCR showed that (S)Rh2 downregulated the transcription of ß-catenin/TCF4 target genes, such as cyclin D1 and c-myc. These results indicated that (S)Rh2 induced cell cycle arrest and apoptosis through the Wnt/ß-catenin signaling pathway, demonstrating its potential as a chemotherapeutic agent for leukemia therapy.

[Ginsenoside Rh2-induced inhibition of histone deacetylase 6 promotes K562 cells autophagy and apoptosis in vivo].

To study the in vivo inhibition effect of ginsenoside Rh2 on humanleukemia cells, and explore its mechanism from autophagy and apoptosis aspects, human leukemia K562 cells allograft tumor models were applied, and after administration of ginsenosides Rh2 by gavage, the tumor diameter, volume and inhibitory rate were measured, and the anti-tumor activity of ginsenosides Rh2 was observed. The levels of HAT and HDAC in tumor tissues were detected by chemical colorimetry assay, and expressions of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5 and HDAC6 were detected by Western blotting assay. The expression levels of vital genes closely associated with autophagy and mRNA expressions of HDAC6 and Hsp90 were detected by Real time-PCR. HE staining was used to observe apoptosis, and immunohistochemistry was used to detect the protein expressions of HDAC6, Hsp90 and activated caspases 3. The results showed that ginsenoside Rh2 could inhibit the growth of k562 cells allograft tumor, with a tumor inhibition rate up to 53.10%. Ginsenoside Rh2 could significantly decrease HDAC activity and decrease the expressions of HDAC1, HDAC2 and HDAC6, and inhibit the expressions of HDAC6 and HSP90, increase the expressions of vital autophagy genes (beclin-1, LC3A and LC3B). Histopathological results showed that ginsenosides Rh2 could significantly increase the tumor apoptosis. Therefore, ginsenoside Rh2 had good anti-tumor effect in vivo, and the mechanism maybe associated with regulating autophagy and apoptosis through HDAC6 and Hsp90 pathways and inhibiting the in vivo proliferation of tumor cells.

Evodiamine Induces Apoptosis and Inhibits Migration of HCT-116 Human Colorectal Cancer Cells.

Evodiamine (EVO) exhibits strong anti-cancer effects. However, the effect of EVO on the human colorectal cancer cell line HCT-116 has not been explored in detail, and its underlying molecular mechanisms remain unknown. In the present study, cell viability was assessed by Cell Counting Kit-8 (CCK-8). Cell cycle and apoptosis were measured by flow cytometry, and morphological changes in the nucleus were examined by fluorescence microscopy and Hoechst staining. Cell motility was detected by Transwell assay. ELISA was used to assess the protein levels of autocrine motility factor (AMF) in the cell supernatant, and protein expression was determined by Western blotting. Our results showed that EVO inhibited the proliferation of HCT-116 cells, caused accumulation of cells in S and G2/M phases, and reduced the levels of the secreted form of AMF. The protein levels of tumor suppressor protein (p53), Bcl-2 Associated X protein (Bax), B cell CLL/lymphoma-2 (Bcl-2), phosphoglucose isomerase (PGI), phosphorylated signal transducers and activators of transcription 3 (p-STAT3) and matrix metalloproteinase 3 (MMP3) were altered in cells treated with EVO. Taken together, our results suggest that EVO modulates the activity of the p53 signaling pathway to induce apoptosis and downregulate MMP3 expression by inactivating the JAK2/STAT3 pathway through the downregulation of PGI to inhibit migration of HCT-116 human colorectal cancer cells.

Down-regulation of phosphoglucose isomerase/autocrine motility factor enhances gensenoside Rh2 pharmacological action on leukemia KG1α cells.

AIMS AND BACKGROUND: Ginsenoside Rh2, which exerts the potent anticancer action both in vitro and in vivo, is one of the most well characterized ginsenosides extracted from ginseng. Although its effects on cancer are significant, the underlying mechanisms remain unknown. In this study, we sought to elucidate possible links between ginsenoside Rh2 and phosphoglucose isomerase/autocrine motility factor (PGI/AMF). METHODS: KG1α, a leukemia cell line highly expressing PGI/AMF was assessed by western blot analysis and reverse transcription- PCR (RT-PCR) assay after transfection of a small interfering (si)-RNA to silence PGI/AMF. The effect of PGI/ AMF on proliferation was measured by typan blue assay and antibody array. A cell counting kit (CCK)-8 and flow cytometry (FCM) were adopted to investigate the effects of Rh2 on PGI/AMF. The relationships between PGI/AMF and Rh2 associated with Akt, mTOR, Raptor, Rag were detected by western blot analysis. RESULTS: KG1α cells expressed PGI/AMF and its down-regulation significantly inhibited proliferation. The antibody array indicated that the probable mechanism was reduced expression of PARP, State1, SAPK/JNK and Erk1/2, while those of PRAS40 and p38 were up-regulated. Silencing of PGI/AMF enhanced the sensibility of KG1α to Rh2 by suppressing the expression of mTOR, Raptor and Akt. CONCLUSION: These results suggested that ginsenoside Rh2 suppressed the proliferation of KG1α, the same as down-regulation of PGI/AMF. Down-regulation of PGI/ AMF enhanced the pharmacological effects of ginsenoside Rh2 on KG1α by reducing Akt/mTOR signaling.