Xiaochaihu Decoction attenuates the vicious circle between the oxidative stress and the ALP inactivation through LPS-catecholamines interactions in gut, liver and brain during CCI₄+ethanol-induced mouse HCC

This study was approved by Guangdong Science and Technology Committee and Guangdong Management Committee for Medical Experimental Animals.

Chinese Kun Ming (KM) mouse were obtained from the Laboratory Animal Research Center of Guangzhou University of Traditional Chinese Medicine. The animals were housed in a plastic cage in an air-conditioned room at 20 ± 2°C with a humidity 58% ± 5% in a 12-h light and dark cycle with free access to standard rat food and tap water.

A total of 80 male KM mice (cleaning grade, 30 days of age, 18-22 g) were used. The KM mouse were divided into four groups, normal group (10 mice), Hepatocellular carcinoma group (HCC,30 mice), were induced as previously described [10] with some minor modifications by subcutaneously injected with 25% CCI₄ olive oil solution (5 mL/kg twice per week) and allowed free access to a 8% ethanol solution as drinking fluid for 4 weeks, and allowed free access to a 0.5% CCI₄-8% ethanol solution as drinking fluid for 20 weeks; HCC + LDSD group (HCC + LDSD, 30 mice), on the basis of HCC model, the mouse were stimulated with the factor of Liver Depression with Spleen Deficiency (LDSD): squeezing tails (30 min/day) for 4 weeks, solitary breeding and intermittent fasting for 20 weeks; HCC + LDSD + XCHD group (XCHD, 30 mice), XCHD was administered by gavage for 8 weeks after 4 weeks of the HCC + LDSD. We recommend 30 mice/model group, this relatively large number of mice is recommended to ensure that enough mice for good statistical analysis, tumor models process often leads to the incidental death of mice.

The XCHD consisting of seven herbal medicines (Table 1) and each crude drug was purchased from Hexiang Pharmaceutical Co., Ltd., China. The origin of materials was confirmed and standardized taxonomically by the Traditional Chinese Medicine Herbal Pharmacy, Guangzhou University of TCM. A decoction of XCHD was prepared in our laboratory (Table 1) from a mixture of chopped crude herbs, extracted in distilled water at 100°C, this mixture was then concentrated to a final concentration of 100 crude drug gram per 100 milliliter, was administered by gavage, 8 g/kg per day for 8 weeks, was dried out (3.34 g) in hot (100°C) water bath for quantitative assay of saikosaponin a of the major constituents in XCHD, the procedure is as follows.

Freeze-dried XCHD were prepared with 70% methanol containing 0.1 N sulfate acid, heated at 100°C for 3 h, Saikosaponin a were converted completely into saikosaponin b by mild acid treatment, the absorbance of Saikosaponin b was detected by UV-spectrophotometer at 252 nm. The amount of Saikosaponin a in XCHD was quantified using the calibration curves of the above-mentioned Saikosaponin a.

The stabilities of Saikosaponin a was examined for 6 days (0, 1, 2, 3, 4 and 5 day respectively) by three sample solution.

Every mouse were autopsied as quickly as possible after natural death and killed, and photographed, and liver were embedded in paraffin and stained with hematoxylin and eosin. The extent of macroscopically apparent inflammation ulceration and tissue injury was done unblinded by two examiners using a scoring system. This tissue was weighed, processed and the resulting supernatant stored at −20°C for the subsequent determination of ONOO⁻, MDA, VMA, ALP-A, for the assay of ALP-C or for the assay of LPS-P.

Intestine macroscopic damage score was calculated as previously described [13] with some minor modifications and graded as 0 = no apparent damage, 1 = slight thickening of intestinal wall, redness, 2 = thickening and adhesions, no apparent ulcers, 3 = adhesions and one or more pin-point ulcers, 4 = adhesions and pin-point ulcers with one or more linear ulcer < 1 cm in length, 5 = one or more linear ulcer > 1 cm in length.

Fecal loading in the cecum was graded as 0 = no fecal loading in the cecum, 1 = mild fecal loading in the cecum, 2 = moderate fecal loading in the cecum, 3 = marked fecal loading in the cecum.

Iiver macroscopic damage score was graded as 0 = reddish brown in color, soft in consistency, smooth surface, 1 = the liver were enlarged, friable, soft in consistency, 2 = hepatic hyperemia, 3 = an enlarged liver with white focal areas of necrosis, 4 = an enlarged liver with white multifocal areas of necrosis, 5 = hepatic steatosis, the liver were soft, yellow, greasy, enlarged, 6 = the liver is mottled red with bile stained areas, of normal or increased size, 7 = contains visible nodules and fibrosis, 8 = micronodular, yellow, fatty, enlarged, 9 = macronodular, brown, non-greasy, shrunken, cirrhosis, 10 = explanted liver showing small single granules bodies, 11 = explanted liver showing large single granules bodies, 12 = explanted liver showing many small granules bodies, 13 = explanted liver showing many large granules bodies.

Extracts from tissue were prepared by homogenization in 9 g/L NaCl at 4°C using a DY89-1 tissue homogenizer. Tissue homogenate was centrifuged at 10000 r/min for 30 min. Clear supernatant were used to determine the ALP-A and ALP-C by lectin (ConA) affinity precipitation method, the precipitation procedure was performed as previously described [14, 15]. The clear supernatant were mixed with 400 μL of an aqueous solution of Con A-Sepharose 6B (5 g/L in distilled water). The mixture was incubated for 30 min at 37°C and centrifuged at 2000 r/min for 15 min. Without disturbing the precipitate, we removed the supernate and measured its ALP-A. ALP-C was calculated by subtracting the corrected value from the total ALP-A, ALP-A was measured with β-glycerophosphate as substrate.

The tissue were initially deproteinized with Somogyi reagent [16] (using 2.0 ml 55 mmol/L NaOH followed by 2.0 ml 75 mmol/L ZnSO₄), after centrifugation, aliquots of supernatant ONOO⁻ concentration was measured from its absorption at 302 nm by using ϵ₃₀₂ = 1670 mol•L^-1•cm^-1[17]. Results were expressed as nmol/L per gram wet tissue.

Samples were weighed and immediately frozen and stored at −20°C until analysis within 2 wk. Samples were subsequently homogenized in buffer and assayed for MDA content using the thiobarbituric acid (TBA) reaction, as described by Uchiyama and Mihara [18]. with some minor modifications. Briefly, 0.5 mL of homogenate (10% concentration) was mixed with 3 mL of 0.05 N HCL. After addition of 1 mL of 0.67% TBA reagent, the tubes were heated in boiling water for 45 min. The color was formed with 4 mL of n-butanol and centrifuged. The color intensity of the butanol layer was estimated by spectrophotometric absorbance at 535 nm. Results were expressed as pmol/L per gram wet tissue.

The protocol was performed as previously described [19, 20] with the following modifications. Lipopolysaccharide was extracted by the hot phenol/water procedure. The polysaccharide moiety was separated from the lipid A by hydrolysis of lipopolysaccharide in 10% acetic acid at 100°C for 2.5-4 h. After in separation of lipid A by centrifugation at 1300 x g for 15 min, the lipid A was washed twice with warm water (70-80°C) and once with acetone. Degraded polysaccharide, obtained from the supernatant fluid, was purified by centrifugation (10500 × g, 3 h) and the supernatant was lyophilized, the phosphate content of LPS was determined with phosphomolybdate-blue spectrophotometry. The amount of inorganic phosphate was quantified using the phosphate standard curve, which was obtained by using serial dilutions of 1.00 mM KH₂PO₄ standard solution.

The protocol was performed as previously described [21] with the following modifications. VMA is extracted by ethyl acetate, is oxidised with periodic acid to vanillin, and the coloured hydrazone formed from the oxidation product and added 2,4-dinitrophenylhydrazine is measured in alkali at 460 nm. The amount of VMA was quantified using the vanillin standard curve, which was obtained by using serial dilutions of 250 ug/ml vanillin standard solution.

The linearity of the absorbance at 252 nm (x) versus concentration (y, μg/mL) curve for Saikosaponin a was used to calculate the contents of the main components Saikosaponin a in XCHD. The correlation coefficients(R) of the calibration curves were 0.9985. The regression equations were Y = 369.82 × -2.071 for Saikosaponin a. These results showed that the calibration curve was a good linearity.

The stability test of Saikosaponin a was evaluated using the sample solution for 6 days. In Table 2, sample solution retained a content of 76.92-100.88% as compared with the initial content at 0 day. The RSD values of contents about saikosaponin a compounds in sample solution were with in 15.52%.

During chemical induction of hepatic cancer by CCI₄ + ethanol, Histological examination of liver sections by H&E staining showed that the induction of hepatic cancer involves four stages: stage 1, hepatic steatosis occurred at 5 to 8 weeks during hepatic carcinoma induction; stage 2, hepatic cirrhosis occurred at 9 to 12 weeks during hepatic carcinoma induction; stage 3, precancerous Lesion of Liver occurred at 13 to 16 weeks during hepatic carcinoma induction; stage 4, hepatic carcinoma occurred at 17 to 24 weeks during hepatic carcinoma induction(Figure 1).

HCC group were significantly more deep red color of liver, the surface of the liver is granular and nodular, a more disordered structure of hepatic lobule, the hepatic cell nuclear anachromasis with nuclear atypia in the H&E stained section. HCC + LDSD group were significantly more dark red color of liver, the surface of the liver is granular and nodular, a more disordered structure of hepatic lobule, the hepatic cell nuclear anachromasis with nuclear atypia in the H&E stained section.

Significantly more injuries in small intestine, cecum, liver(mainly hyperplasia) of HCC, only a slight non-significant reduction was observed in brain weights, compared with normal group, the most in HCC + LDSD group, compared with HCC group, after treatment with XCHD, all of which were improved, compared with HCC + LDSD group. The other organs do not show obvious macroscopic damage (Table 3).

ONOO⁻, MDA, VMA, LPS-P, ALP-C were increased, ALP-A were decreased in the gut, liver and brain of HCC group, compared with normal group, the most in HCC + LDSD group, compared with HCC group, after treatment with XCHD, all of which were improved, compared with HCC + LDSD group (Table 4).

Correlative analysis showed that there was a positive association between gut-liver-brain injury and ONOO⁻, MDA, VMA, LPS-P, ALP-C, between ONOO⁻, MDA, VMA, LPS-P and ALP-C in the gut, liver and brain, there was a negative association between gut-liver-brain injury and ALP-A, between ALP-A and ONOO⁻, MDA, VMA, LPS-P, ALP-C in the gut, liver and brain (Tables 5, 6, 7 and 8).