br Sigma g glycerol Chemworks Beijing China g propylene glyc
(Sigma), 1.5 g glycerol (Chemworks, Beijing, China), 1.5 g propylene glycol (Xilong Chemical Co. Ltd., Guangxi, China), and sterile water were churned and heated to 60 °C. The water phase was added slowly into the oil phase and expanded to 10 mL with sterile water to form the colostrum. Then, the colostrum was shaken with a DS-5510DT ultra-sonic instrument (Shengxi, Ultrasound equipment Ltd., Shanghai, China) for 10 min to form the emulsion. The concentrations of high-dose, middle-dose, and low-dose emulsions were 2.5, 0.25, and 0.05 mg/mL, respectively. All emulsions filtered through 0.22 μm membrane filter and stored at 4 °C.
2.6. Establishment of LNM models
The experimental protocol was approved by the ethics review committee for animal experimentation at our institution. All experi-mental mice were raised in special pathogen free (SPF) condition in the Experimental Animal Center of PLA 302 Hospital (Beijing, China). Six-week-old female BALB/c nude mice were anesthetized by ether in-halation anesthesia and then placed in a supine position. The human CRC lymphatic metastasis model of the nude mouse was established by injecting 4 × 106 HCT116 SP-13786 at a total volume of 40 μL sub-cutaneously into the right hind footpad, similar to previous reports [25,26].
2.7. Eﬀect of HELT fraction emulsions on in situ tumor and lymph metastasis
Cinobufacini Injection (CI) is an aqueous extraction from toad skin, which is now widely used in clinical therapy for various cancers, ar-rhythmia, and heart diseases in China. In the present study, we took CI as a positive control. At the 3rd day of injection, 48 tumor-bearing nude mice were randomized into 6 groups and treated with normal saline (NS) (blank control), control emulsion (CE) containing 1.25% (v/v) DMSO, CI (0.4 mL/kg equals clinical therapeutically dose: intravenous injection of 20 mL/50 kg per day for adults, equivalent to 0.5 g/mL of herbal pieces prepared for decoction; Jinchan Biotech, Anhui, China), F18 high-dose emulsion (HD-E; 5 mg/kg), F18 middle-dose emulsion (MD-E; 0.5 mg/kg), and F18 low-dose emulsion (LD-E; 0.1 mg/kg). Each group underwent footpad peritumoral injection (lymphatic che-motherapy) at a dosage of 0.04 mL/20 g for 18 days. The gross tumor volume was calculated according to the following equation: V (mm3) = (length × width2) / 2. After treatment, the mice were killed. The footpad tumors, subiliac lymph nodes, medial iliac lymph nodes, and renal lymph nodes were dissected and observed by hematoxylin and eosin (H&E) staining. The tumor inhibition rate (IR) and LNM rate were respectively calculated according to the following equations: IR = [1 −tumor weight (experimental) / tumor weight (con-trol)] × 100%; LNM rate = [1 −number (metastatic) / number (de-tected)] × 100%.
2.8. Eﬀect of HELT fraction emulsions on immune function
The spleen is an important immune organ, and the spleen index (SI) can reflect the immune state to a certain extent. Thus, the spleen was removed after treatment and the SI value was calculated by (spleen weight / body weight) × 10. Then spleen cells were cultured according to the standard procedures. Since the lack of thymus and the loss of T cell immunity, natural killer (NK) cells and B cells become the main force of antitumor immunity in nude mice. The activated NK cells se-crete IFN-γ while eﬀector B cells synthetizes and secretes antibodies. Thus, we respectively analyzed the proportion of NK cells among per-ipheral blood and spleen by Flow Cytometry (FCM). In addition, we respectively detected the content of IFN-γ and IgG1 in serum as well as the splenic cell supernatant by enzyme-linked immune sorbent assay (ELISA) according to the manufacturer's instructions. International Immunopharmacology 70 (2019) 241–251
2.9. Evaluation of the potential toxicity
Potential toxicity treated with HELT faction emulsions at diﬀerent concentrations has been investigated. Gross measures such as weight loss, behavior and feeding were evaluated. The blood was drawn after treatment and the biochemical indexes of blood and liver were ana-lyzed.
2.10. Statistical analysis
Data were expressed as the mean ± SD. Diﬀerences between groups were analyzed by one-way analysis of variance (ANOVA) and Student t-test using the SPSS 20.0 statistical software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant diﬀerence.
3.1. Physical property and eﬀects on cell growth of fractions of bufadienolides
The physical property of 22 fractions of bufadienolides was shown in Table 1. F1–F5 were water-soluble. F6–F22 were soluble in DMSO, an eﬀective solvent for a wide array of organic materials.