ABSTRACT
Objective: He-Wei Granule (HWKL) is a modern product derived from the modified formulation of traditional Chinese medicine Banxia Xiexin Decoction (BXD), which remarkedly enhanced the anti-proliferation activity of cyclophosphamide (CTX) on HepG2 and SGC-7901 cell lines in vitro in our previous research. The aim of the study was to investigate the synergistic effects of HWKL and CTX using a transplanted H22 hepatocellular carcinoma mouse model. Methods: The CTX-toxic-reducing efficacy of HWKL was evaluated by hematology indexes, organ indexes and marrow DNA detection. To investigate the underlying mechanisms, histopathology test, immunohistochemistry test and TUNEL staining were conducted. The efficacy of HWKL on the micro-vessel density (MVD) in tumor tissue was also evaluated by measuring CD34 level. Results: High dose HWKL (6.75 g/kg) markedly attenuated CTX-induced hepatotoxicity and myelosuppression while significantly enhanced CTX anticancer efficacy in vivo. Further mechanism investigation suggested that high dose HWKL significantly increased cleaved Caspase 3 level and promoted apoptosis in tumor tissue by up-regulating Bax expression and down-regulating Bcl-2 and FasL expressions. Compared with CTX alone group, the decrease in LC-3B and Beclin 1 levels suggested that the autophagy in H22 carcinoma was significantly inhibited with addition of high dose HWKL. ELISA assay results indicated that the autophagy inhibition was achieved by decreasing p53 expression, blocking PI3K/AKT/mTOR pathway and recovering Th1/Th2 cytokine balance. In addition, CD34 and EGFR immunohistochemistry assay suggest that high dose HWKL could significantly decrease micro-vessel density (MVD) and inhibit angiogenesis in H22 carcinoma. Conclusion: It can be concluded that high-dose HWKL enhanced CTX efficacy by promoting apoptosis, inhibiting autophagy and angiogenesis in tumor tissue while significantly alleviated CTX-induced toxicity, and could be applied along with CTX in clinical treatment as a supplement agent.
ABSTRACT
OBJECTIVE: To study the effects of Wuzhi capsule/schisantherin A (SchA) combined with cyclophosphamide on the pharmacokinetics of cyclophosphamide (CTX) in rats. METHODS: A total of 36 rats were randomly divided into CTX group (via tail vein, iv, CTX solution 300 mg/kg), CTX+WZC group (ig, Wuzhi capsule 300 mg/kg+via tail vein, iv, CTX solution 300 mg/kg), CTX + SchA low-dose, medium-dose, high-dose and excessive high-dose groups (ig, SchA 30, 300, 3 000, 30 000 μg/kg+via tail vein, iv, CTX solution 300 mg/kg) with 6 rats in each group. Blood samples were collected from orbital venous plexus of rats before medication and 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48 h after medication.UPLC-MS/MS method was applied for concentration determination of CTX and its metabolites [de-chloroethyl CTX (DC-CTX), 4-ketone CTX (4-keto CTX), carboxyl phosphamide (CPM)] in plasma of rats. The plasma concentration-time curve was obtained. The pharmacokinetic parameters were fitted by using DAS 2. 0 software. RESULTS: The maximum plasma concentration (cmax) of DC-CTX in CTX group, CTX+WZC group, CTX+SchA low-dose, medium-dose, high-dose and excessive high-dose groups were (22 167. 85 ±2 844. 93), (10 920. 53 ± 1 490. 89), (18 951. 29 ± 1 558. 81), (18 622. 08 ± 791. 19), (18 515. 20 ± 2 560. 61), (15 133. 21 ± 1 305. 07) μg/mL, respectively; the area under the curves (AUCo-48 h) were (173 864. 01 ± 65 342. 21), (100 996. 98 ± 33 530. 02), (137 028. 16 ± 45 975. 19), (131 650. 18 ± 53 196. 41), (113 699. 40 ± 34 131. 36), (110 773. 27 ± 30 307. 15) μg·mL/h, respectively. Compared with CTX group, cmax of DC-CTX in CTX group, CTX+SchA low-dose, medium-dose, high-dose and excessive high-dose groups were decreased by 50. 74％, 14. 51％, 16. 10％, 16. 48％, 31. 73％, respectively. AUC0-48 h were decreased by about 42. 23％, 21. 45％, 24. 63％, 33. 37％, 36. 55％, respectively; with statistical significance (P<0. 05). The pharmacokinetic indexes as t1/2, tmax had no significant change. CONCLUSIONS: To some degree, both WZC and SchA can reduce the generation of DC-CTX, which indicates both of them can inhibit CTX toxicity metabolism pathway so as to reduce the generation of toxic metabolite chloroacetaldehyde. The inhibitory effect of SchA on toxicity metabolism pathway is weaker than that of WZC, and does not have a dose-dependent inhibitory effect.