| Non-small cell lung cancer(NSCLC)is one of the main threats to human health Immunotherapy and targeted therapy have become important research directions of anti-cancer drugs for NSCLC in recent years.Tumor-derived exosomes(TEXs)can promote tumor invasion,metastasis,angiogenesis,and immune escape,etc.And the TEX has become a hot topic in cancer research.The biosynthesis and function of TEXs are closely related to cell membrane lipid rafts.Solanum lyralum Thunb.,as a traditional anti-cancer Chinese medicine,has the effects of heat-clearing,detoxifying,and removing dampness and swelling,and is widely used in the clinical treatment of various tumors.Solanum lyratum is rich in different types of compounds,and the alkaloids in Solanum lyratum have excellent antitumor efficacy.In a former study,we found that the total alkaloids from Solanum lyratum(TAS)had significant inhibitory effects on NSCLC tumorigenesis in vitro and in vivo.Pharmacological studies have shown that TAS can inhibit tumor angiogenesis.Previous studies have shown that TAS contains a variety of steroidal alkaloids and proved that steroidal alkaloids contained in TAS can change the morphology and function of cell membrane lipid rafts by agglutinating cell membrane cholesterol in tumor-derived vascular endothelial cells(Td-ECs)The purpose of this study is to make further research on the anti-tumor effect of TAS and its material basis on the foundation of the previous studies.Combining the close relationship between TEXs and cell membrane lipid rafts,we studied the effects of alkaloids from Solanum lyratum on TEXs in vitro and in vivo.Similar to steroid glycoalkaloids,saponins also have the property of binding to cholesterol and the activity of destroying the integrity of lipid rafts.Combining the above facts,we proposed a research hypothesis that steroidal glycoalkaloids and saponins can affect the generation and function of TEXs by disrupting the integrity of the cell membrane lipid rafts.In addition,we took ginsenoside Rg3 as an example to conduct a preliminary study on the effect of saponins on the generation and function of TEXs,to perform preliminary verification of the above hypothesis.Besides,the Antexiao capsule(AtxC)is a new anti-tumor Chinese medicine with TAS as a raw material.In this paper,a systematic study of the preclinical safety of AtxC is conducted in order to provide safety data for the development and application of AtxCThe main research methods are as follows:1.To study the chemical composition of TAS,a variety of chromatographic techniques were used to separate and purify the components of TAS.The structures of the isolated monomer compounds were identified by mass spectrometry,1D and 2D-NMR.2.To study the anti-tumor activity of TAS and its chemical constituents,the effects of isolated monomeric compounds 1-7 and 11-13 on the proliferation of Td-EC cells were tested by the MTT method.A scratch wound assay was used to investigate the tumor angiogenesis inhibitory activities of compounds 1-7 and 11-13 on Td-EC cells.A mouse transplanted tumor model of S180 sarcoma was used to investigate the anti-tumor activity of the high polar region of TAS(TASG)and the low polar region of TAS(TASS).The low-,medium-and high-dose groups administered TASS or TASG in the dose of 50 mg·kg-1,100 mg·kg-1,and 200 mg·kg-1,respectively.TASS or TASG were administered intragastrically for 10 days.TAS and cyclophosphamide(CTX)were used as positive control for the antitumor effect of TASS and TASG,and the thymus and spleen index of the mice were measured.Enzyme-linked immunosorbent assay(ELISA)method was used to detect the levels of cytokines interleukin-6(IL-6),transforming growth factor beta(TGF-?)and interferon gamma(IFN-y)in tumor-bearing mice in the TAS group,to investigate the effects of TAS on the immune system of tumor-bearing mice.3.To investigate whether the inhibitory effects of glycoalkaloids from Solanum lyratum and ginsenoside Rg3 on tumors are related to TEXs,we used exosomes isolation kit and ultracentrifugation to isolate exosomes derived from A549 cells TEXs were identified by Western blotting,transmission electron microscopy,and nano-particle size analysis.The effect of A549-derived exosomes on the angiogenesis of human umbilical vein vascular endothelial cells(HUVECs)was examined by an in vitro scratch wound healing assay,tube-formation assay,and Transwell chamber inserts invasion experiment.The effects of TAS,SA1 and ginsenoside Rg3 on the proliferation of A549 cells were tested by the MTT method.LDH leakage test was used to detect the effect of SA1 and ginsenoside Rg3 on the membrane integrity of A549 cells.After incubating A549 cells with TAS at a final concentration of 10?g-mL-1 and SA1 and ginsenoside Rg3 at final concentrations of 10 ?M for 48 h,the size of the A549-derived exosomeswas measured.And the isolated A549-derived exosomes were applied to HUVECs,to investigate the effects of TEXs secreted by A549 cells incubated with alkaloids from Solanum lyratum or ginsenoside Rg3 on the angiogenesis of HUVECs.Proteomics was used to detect the changes of the proteome in SA1 intervention and non-intervention A549 cells and exosomes secreted by them.A nude mouse model of A549 cell xenograft tumor was established using the BALB/c nude mice.A549 tumor-bearing nude mice were administered intratumorally with a single dose(100 ?g)of A549-derived exosomes(Control exosome)or exosomes secreted by SA1-treated A549 cells(SA1 exosome).The frequency of administration was maintained twice a week.Paclitaxel was used as positive control(Taxol,8 mg·kg-1,intraperitoneal injection),and a vehicle control group(Model)was set.The tumor growth status was dynamically observed.Animals were sacrificed 28 days after the treatment.The tumor masses were removed and weighed.To investigate the effects of exosomes,which secreted by A549 cells or A549 cells after SA1 intervention,on the tumor growth in vivo4.To investigate the preclinical safety of AtxC,we used a single intragastric administration of the contents of AtxC in mice and beagle dogs,and observed the effects of AtxC on coordinated movement,autonomous activity,and the sleep-inducing effects with a subthreshold dose of sodium pentobarbital in mice,and on electrocardiogram(ECG),blood pressure and respiratory parameters in beagle dogs.The contents of AtxC in rats and beagle dogs were administered orally by single gavage.The body weight,food intake,ECG and other indicators were observed to investigate the acute toxicity of AtxC.The contents of AtxC were orally administered to rats repeatedly for 26 weeks and to beagle dogs repeatedly for 39 weeks.The body weight,ECG,and clinical-pathological characteristics were used to observe the long-term systemic toxicity of AtxC.The main research results are as follows1.A total of 14 steroidal glycoalkaloids were isolated and identified,including 12 new compounds(1-12)and two known ones(13-14).The structures of the 14 isolated monomers were identified as:(3?,5a,20S,22S,23R,25S)-16,22-epoxy-23,26-imino-cholestan-3-ylO-?-D-glucopyranosyl-(1?2)-O-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(1),(3?,20S,22S,23R,25S)-16,22-epoxy-23,26-imino-cholestan-5-en-3-ylO-?-D-glucopyranosyl-(1?2)-O-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(2),16,23-epoxy-22,26-imino-cholestan-5,22(N),23,25(26)-tetraene-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-glucopyranosyl-(1?4)-?-D-galac topyranoside(3),(3?,25R)-16,23-epoxy-23,24-imino-cholestan-5,16,20,23(N)-tetraene-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-glucopyranosyl-(1?4)-?-D-galac topyranoside(4),(3?,5?,25R)-16,23-epoxy-23,24-imino-cholestan-16,20,23(N)-triene-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-glucopyranosyl-(1?4)-?-D-galacto pyranoside(5),15?-hydroxyl-(3?,5?,25R)-16,23-epoxy-23,24-imino-cholestan-16,20,23(N)-triene-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-glucopyranosyl-(1?4)-/8-?-D-galactopyranoside(6),15/8-hydroxyl-(3?,25R)-16,23-epoxy-23,24-imino-cholestan-5,16,20,23(N)-tetraene-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(7),15?-ethoxy-(3?,25R)-16,23-epoxy-23,24-imino-cholestan-5,16,20,23(N)-tetraene-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-glucopy ranosyl-(1?4)-?-D-galactopyranoside(8),15?-ethoxy-(3?,5?,25R)-16,23-epoxy-23,24-imino-cholestan-16,20,23(N)-triene-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-gl ucopyranosyl-(1?4)-?-D-galactopyranoside(9),15?-hydroxyl-(3?,5?,25R)-16,23-epoxy-23,24-imino-cholestan-16,20,23(N)-triene-3?-ol-3-O-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(10),(3?,1 6R,20R,22a,25R)-spirosolan-5-en-3-ylO-?-D-glucopyranosyl-(1?2)-O-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(11),(3?,5?,16R,20R,22?,25R)-spirosolan-3-ylO-?-D-glucopyranosyl-(1?2)-O-?-D-gluco pyranosyl-(1?4)-?-D-galactopyranoside(12),16,23-epoxy-22,26-imino-cholest-22(N),23,25(26)-trien-3?-ol-3-O-?-D-glucopyranosyl-(1?2)-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(13),(3?,22?,25R)-spirosolan-5-en-3-ylO-?-D-glucopyranosyl-(1?2)-O-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(14,SA1).2.When compounds 1-7 and 11-13 interfered with Td-EC cells at the highest concentration of 100 ?M for 48 h,the inhibition rates of proliferation did not reach 50%.Compounds 1-6 and 11-13 at 25 ?M can significantly inhibit the migration of Td-EC cells(P<0.05).The vascular endothelial growth factor(VEGF)significantly promoted the migration of Td-ECs(P<0.05),while compounds 1-7 and 11-13 all inhibited the migration ability of Td-ECs induced by VEGF in a dose-dependent manner(P<0.05).Among all the Solanum lyratum administration groups,the tumor inhibition rate of S180 tumor-bearing mice was higher in the TASS-L group,the TASG-H group,and the TAS group,which were 39.71%,43.92%,and 42.13%,respectively.Compared with the model group,the thymus and spleen indices of the S180 tumor-bearing mice in the CTX group were significantly reduced(P<0.01).The spleen indices of the animals in all groups of Solanum lyratum alkaloids except the TASG-H group were all increased,while the spleen indices of the TASS-L group and TASS-H group both increased significantly(P<0.01,P<0.05).Compared with the CTX group,the thymus indices of the animals in the TASS-M,TASS-H,and TASG-H groups and the spleen ndices of the animals in all groups of Solanum lyratum alkaloids were significantly increased.Compared with the model group and the CTX group,the levels of IFN-y in the serum of the tumor-bearing animals in the TAS group were significantly increased(P<0.05).The above results show that TAS and its high polar region as well as its low polar region can inhibit sarcoma growth in S180 tumor-bearing mice,and can improve the immune function of S180 tumor-bearing mice.3.Although SA1 and ginsenoside Rg3 have a weak inhibitory effect on the proliferation of A549 cells,they have a destructive effect on the integrity of A549 cell membranes and could increase the LDH leakage rate of A549 cells in a dose-dependent manner(P<0.05).The isolated A549-derived exosomes were round or oval membrane vesicles in shape.The expression of the exosome marker proteins CD9 and CD63 in of exosomes secreted by A549 cells was positive,and with the extension of the culture time,the diameter of exosomes increased.Compared with the blank control group,TEXs(Control exosome)secreted by A549 cells without intervention significantly promoted the scratch healing of HUVECs cells(P<0.01).Compared with the blank control group and the Control exosome group,exosomes secreted by A549 cells after TAS and SA1 intervention significantly inhibited the scratch healing of HUVECs.And exosomes secreted by A549 cells after TAS and SA1 intervention significantly inhibited the effect of A549-derived exosomes on promoting scratch healing of HUVECs(P<0.01).Exosomes extracted from A549 cells that had been incubated with SA1 or Rg3 significantly inhibited the invasion ability and tube formation of HUVECs.The particle size of TEXs was mainly concentrated in the range of 60nm to 90 nm,and the concentrations of TEXs in the supernatant of A549 cells treated with TAS,SA1,or Rg3 increased by 10,3.8,and 3.7 times compared to the control exosomes.Proteomics results showed that there were 1,154 differential proteins between SA1 intervention and non-intervention A549 cells.Their major functions involved Poly(A)RNA binding,and these differential proteins were associated with biological processes such as translation and rRNA processing.There are 746 differential proteins between SA1 intervention and non-intervention A549-derived exosomes.Their major functions involved Poly(A)RNA binding,protein binding,and other functions,and these differential proteins were associated with biological processes such as cell adhesion and mRNA splicing.Differential proteins detected by proteomics were related to more than 40 metabolic pathways,500 signaling pathways and 20 protein-protein interaction networks.The results of proteomics research provided multiple targets for subsequent research.Compared with the model group and the control exosome group,the SA1 exosome significantly reduced the tumor volume and tumor weight of tumor-bearing nude mice(P<0.01).The tumor inhibition rate of SA1 exosome group was 70.48%,and the control exosome also showed a certain inhibitory effect on tumor growth compared with the model group.4.A single intragastric administration of AtxC did not affect the autonomic activity and coordinated movement of mice,nor did it affect the ECG,blood pressure,breathing and other indicators of beagle dogs.But AtxC could increase the sleeping rate induced by a subthreshold dose of pentobarbital sodium at a single dose of 800 mg·kg-1(P<0.01).The maximum tolerance to single-dose in rats of AtxC was 4.0 g·kg-1,in dogs was estimated to be over 6.0 g·kg-1.And AtxC administered in a single gavage dose of 16.0 g·kg-1 could reduce the body weight gain and food intake(P<0.05)in rats.The maximum tolerance to repeated-dose in rats of AtxC was 0.8 g·kg-1,in dogs was 320 mg·kg-1.AtxC administered in a repeated gavage dose of 2.0 g·kg-1 could cause gastric lesions both in rats and dogs.And AtxC administered in a repeated gavage dose of 800 mg·kg-1 could significantly reduce the body weight gain and serum TP,GLOB and ALB(P<0.05)in dogs.Through the above research,this paper has obtained the following research conclusions.(1)12 new steroidal glycoalkaloids were isolated from TAS in this study.It was proved that most of the isolated monomeric alkaloids from TAS have the activity of inhibiting tumor angiogenesis in vitro.And proved that TAS has anti-tumor effect and can improve the immune function of S180 tumor-bearing mice.(2)It proved that A549-derived exosomes can promote the angiogenesis of HUVECs in vitro.And the intervention of TAS,SA1 or Rg3 on A549 cells makes the exosomes secreted by A549 cells gain significant angiogenesis inhibitory activity in vitro and can promote the number of exosomes secreted by the A549 cells.The intervention of SA1 can also increase the antitumor activity of exosomes secreted by A549 cells in vivo.The effect of SA1 on A549-derived exosomes is related to its ability to change the proteome of A549 cells and A549-derived exosomes.(3)This thesis preliminarily verified the hypothesis that steroidal glycoalkaloids and saponins can affect the generation and function of TEXs by disrupting the integrity of the cell membrane lipid rafts,which provides a new idea for the antitumor research of steroidal glycoalkaloids and saponins.(4)This paper also proved that oral administration of the new anti-tumor Chinese medicine AtxC with TAS as a raw material has good safety. |
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