Mechanism of Simiao Yongan decoction in the treatment of arteriosclerosis obliterans of lower extremity through JAK/STAT signaling pathway

Objective:To predict the effective components,potential targets and pathways of Simiao Yong’an Decoction in the treatment of lower extremity arteriosclerosis obliterans(ASO)based on the method of network pharmacology,and to explore the mechanism of Simiao Yong’an Decoction in treating ASO combined with cell experiment.Methods:TCMSP database was used to screen the active components and targets of Simiao Yong’an decoction,and Genecards and OMIM databases were used to obtain ASO related proteins;PPI network of drug disease target proteins was constructed by string platform;go function and KEGG pathway enrichment of Simiao Yong’an Decoction ASO target were analyzed by David database.The drug target pathway with high correlation with ASO was selected.The model of vascular smooth muscle cell injury(VSMCs)induced by ox LDL was used,and Simiao Yong’an decoction containing pharmaceutical Qing was given to verify the therapeutic effect of Simiao Yong’an Decoction on ox LDL induced VSMCs and its regulation on highly correlated target pathway.Results:A total of 126 active components of Simiao Yong'an Decoction were screened,40 targets of Simiao Yong’an Decoction ASO were selected,99 go biological processes and 48 related signal pathways were related to ASO;the experimental results showed that with the passage of time,Simiao Yongan decoction could significantly inhibit the proliferation of VSMCs(P<0.05);compared with the model group,the percentage of BrdU positive cells in each dose group of Simiao Yong’an decoction was significantly higher than that in the model group In addition,Simiao Yong’an decoction could significantly inhibit the proliferation of VSMCs(P<0.05);in addition,Simiao Yongan decoction could inhibit the levels of IL-6 and IL-1β(P<0.05);significantly inhibit the expression of PCNA,JAK2 and STAT3 mRNA(P<0.05);Conclusions:Simiao Yong’an decoction has the effect of"multi-component,multi-target and multi-channel"in the treatment of ASO.It can inhibit the activation of JAK/STAT signaling pathway,play an anti-inflammatory role and inhibit the proliferation of vascular smooth muscle cells.

Inhibitory effect of M3 receptor antagonist 4-DAMP on melanoma proliferation of A375 cell line

Objective: he effect of M3 receptor antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) on the proliferation of melanoma were explored, and the development prospect and significance of M3 receptor antagonist in the field of anti-tumor were discussed. Methods: The viability of tumor cells was detected by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) colorimetry. The optimal concentration of 4-DAMP was screened and the effect of the drug was confirmed by EdU (5-ethynyl-2'-deoxyuridine) fluorescence staining. Adult male BALB/c nude mice were divided into control group, 5-Fu (5-Fluorouracil, 5-Fluorouracil, 20 mg/kg/d) group and 4-DAMP (2 mg/kg/d) group to establishtumor-bearing model in vivo. The tumor volume curve was calculated and plotted. Mice were killed after 21 days of continuous administration. Strip the tumor, take pictures and meatured the weighs, and calculate the inhibition rate. [1]The spleen was weighed and the spleen index was culculated. H & E staining was used to observe the pathological changes of tumor sections. The gene expression of M3R、MIA (Melanoma inhibitory activity, MIA)、SP-1 (Transcription factor Sp1, SP-1) and Lnc-SPRY4-IT1 (SPRY4 Intronic Transcript 1,Lnc-SPRY4-IT1) in A375 cell line was detected by real-time quantitative PCR (quantitative reverse-transcription polymerase chain reaction, qRT-PCR). The protein level of ERK1/2 (extracellular signal-regulated kinase 1/2,ERK1/2) was detected by Western Blot. Results: 4-DAMP could significantly inhibit the activity of A375 cell line (P < 0.01), and EdU staining demonstrated that 4-DAMP inhibited the growth of A375 cell line. The results of animal experiments showed that compared with the control group, the volume and mass of tumor in 5-Fu group and 4-DAMP group were significantly reduced (P < 0.05) and H & E staining showed that the intercellular space became larger, connective tissue increased, and tumor growth was obstructed. Compared with the control group, the tumor inhibition rate of 4-DAMP group was 72.01% (P < 0.01). There was no significant difference in body mass and spleen index. Compared with the control group, the expression of MIA, SP-1 and LncRNA-SPRY4-IT1 was decreased by 4-DAMP or si-M3R transfection. Compared with the control group, the expression of ERK1/2 was decreased by 4-DAMP or si-M3R transfection. Conclusions: These results suggest that M3 receptor antagonist 4-DAMP can inhibit the proliferation of melanoma and reduce the expression of mRNA of MIA, SP-1 and LncRNA-SPRY4-IT1 and the protein level of ERK1/2 in A375 tumor cells.