Screening Antitumor Fraction From Oxytropis Falcata In Vitro And In Vivo And Its Potential Mechanism Through Regulation Of TLRs/NF-κB Signaling Effectors

Evidence has demonstrated that inflammation plays an important role in tumorgenesis and progression.Oxytropis falcata Bunge(O.falcata)is honored as "the king of herbal medicine" in Tibetan medicine for its prominent activities.Previous studies have revealed that this herb exerts analgesic,anti-inflammatory and antitumor effects in vitro.However,its antitumor mechanism and the relationship between antitumor and anti-inflammation remain unclear.In general,we strive to reveal the molecular mechanism of antitumor effect of O.falcata by exploring the ’interior relations between its anti-inflammatory and antitumor activity.In this experiment,O.falcata was isolated to determine the optimal site of lung cancer activity in vitro and in vivo;the effect of Liposoluble alkaloids on the TLRs/NF-κB signaling pathway was detected by western blot to clarify the molecular mechanism of anti-tumor of O.falcata.1.Extracting O.falcata and screening the active fraction against A549O.falcata was extracted with ethanol and separated into six parts.CCK-8 method was used to screen the active parts for proliferation inhibition of A549 cell line.The result was that all of Liposoluble alkaloids,flavonoid extract,volatile oil and 95%Ethanolic extract had significant inhibiting effects on proliferation of A549 cells and the ICso of Liposoluble alkaloids was minimal which indicated its optimal effect in vitro,providing the scientific basis and reference for further in vivo antitumor experiment.2.Determination of Liposoluble alkaloids and flavonoids in O.falcataIn the experiment,Thermopsine was used as the reference substance and the alkaloid composition of the liposoluble alkaloid of the O.falcata was detected by the acid dye colorimetric method.The result indicated that the concentration of reference substance(20.06 μg·mL-1～100.03 μg·mL-1)had good linear relationship with absorbance.Ltuin was used as the reference substance and the content of total flavonoids of O.falcata a was detected by NaNO2-Al(NO3)3-NaOH colorimetry.The result indicated that the concentration of rutin standard solution(105μg · mL-1～1260μg·mL-1)had good linear relationship with absorbance.Methodological observations showed that both of colorimetrys possessed good precision,stability,repeatability and high recovery rate.3.Exploring acute toxicity and antitumor effect on Lewis lung carcinoma mice The acute toxicity of 95%Ethanolic extract,Liposoluble alkaloids,Flavonoids were assessed was calculated by using Bliss method.The clinical dose of O.falcata is 0.3～0.5g,the maximum tolerable dose of 95%Ethanolic extract was 18000 mg·kg-1,which was 2808 times of the maximum clinical dose.The LD50 of Lipidsoluble alkaloids was 3856.2 mg·kg-1,which was 141567 times of the clinical maximum dose.The LD50 of Flavonoids extract was 6707.5 mg·kg-1,which was 1569 times of the maximum clinical dose,which indicated that the three extracts of O.falcata were safe under the recommended dosage.Lewis lung cancer model was established and C57BL/6 mice were divided into blank control,positive group,model group,95%Ethanolic extract groups(including low,middle,high dosage groups),Liposoluble alkaloids groups(including low,middle,high dosage groups)and Flavonoid groups(including low,middle,high dosage groups).After treatment,inhibitory rate,thymus index and spleen index of mice were calculated;tumor cell apoptosis was detected by flow cytometry;morphological status of cells were observed by HE staining.It turned out that all of the three extracts of O.falcata had antitumor effect,with Liposoluble alkaloids proven as the best.Compared to the blank group,all groups of Liposoluble alkaloids had significant difference(P<0.01 or P<0.05)which inhibitory rate of 40.2%in high dose group.The positive immune index was significantly decreased(P<0.01)compared with the blank group,but there was no significant difference between the three extacts of O.falcata.Both of these parts could induce Lewis Lung cancer cell apoptosis and the rate raised according to drug concentrations.Apoptosis ratio in alkaloid groups was higher than model group,with high dose group of which reached 55.3%.Pathological section showed that each group had different necrosis of cancer cells,hyperplasia of fibrous tissue,and large necrotic areas in tumor cells of Lipidsoluble alkaloid group.The results demonstrated that the anti-tumor mechanism of Lipidsoluble alkaloid was different from the cytotoxicity of cyclophosphamide,and the Liposoluble alkaloids of O.falcata could induce Lewis lung cancer cell apoptosis with no immunosuppressive effect.4.Exploring the effect of Liposoluble alkaloids on TLRs/NF-κB signaling pathway of mice lung cancer cellsThis section is to inspect the activity against Lewis lung tumour cancer in vivo of 95%Ethanolic extract,Liposoluble alkaloids,Flavonoids of O.falcata,which are selected from the examination of the activity agaist A549 lung cancer cell in vitro.Liposoluble alkaloids showed the optimal activity.After the Liposoluble alkaloids intervention,the tumor tissue of tumor-bearing mice treated with liposoluble alkaloid was quantitatively extracted,and the expressions of TLR4,MyD88,NF-κB,TRAF-6 were detected by WB.Compared with model group,Liposoluble alkaloids significantly reduced the expression of these proteins,which indicated that O.Falcata inhibited the expression of NF-κB by reducing the expression of TLR4,then inhibiting the growth of tumour by controlling downstream factors on NF-κB pathway and transcription of apoptosis related cytokine growth.