Effect of JIANPI HUOXUE decoction on inflammatory cytokine secretion pathway in rat liver with lipopolysaccharide challenge

AIM： To evaluate the effect of Chinese traditional medicinal prescription, JIANPI HUOXUE decoction （JHD） on cytokine secretion pathway in rat liver induced by lipopolysaccharide （LPS）. METHODS： Twenty-four male SD rats were divided into normal group （n = 4）, model group （n = 10） and JHD group （n = 10） randomly. Rats in model group and JHD group were administrated with normal saline or JHD via gastrogavage respectively twice a day for 3 d. One hour after the last administration, rats were injected with LPS via tail vein, 50 μg/kg. Simultaneously, rats in normal group were injected with equivalent normal saline. After LPS stimulation for 1.5 h, serum and liver tissue were collected. Pathological change of liver tissues was observed through hematoxylineosin （H.E.） staining. Tumor necrosis factor alpha （TNF-α） in serum were assayed by enzyme linked immunosorbent assay （ELISA）. The protein expression of TNF-α, phosphorylated inhibit-κB （p-κB） and CD68 in liver were assayed by Western blot. The distribution of CD68 protein in liver was observed through immunohistochemical staining. The mRNA expression of TNF-α, interleukin-6 （IL-6）, CD14, toll-like receptor 2 （TLR2） and TLR4 in liver were assayed by real-time RT-PCR.RESULTS： Predominant microvesicular change, hepatocyte tumefaction and cytoplasm dilution were observed in liver tissues after LPS administration as well as obvious CD68 positive staining in hepatic sinusoidal. After LPS stimulation, serum TNF-α （31.35 ± 6.06 vs 12225.40 ± 9007.03, P 〈 0.05）, protein expression of CD68 （1.13 ± 0.49 vs 3.36 ±1.69, P 〈 0.05）, p-IκB （0.01 ±0.01 vs 2.07 ＋ 0.83, P 〈 0.01） and TNF-α （0.27 ± 0.13 vs 1.29 ± 0.37, P 〈 0.01） in liver and mRNA expression of TNF-α （1.96 ± 2.23 vs 21.45 ±6.00, P 〈 0.01）, IL-6 （4.80 ± 6.42 vs 193.50 ± 36.36, P 〈 0.01） and TLR2 （1.44 ± 0.62 vs 4.16 ± 0.08, P 〈 0.01） in liver were also increased significantly. These pathological changes were all improved in .1HD group. On the other hand, TLR4 mRNA （1.22 ± 0.30 vs 0.50 ± 0.15, P 〈 0.05） was down-regulated and CD14 mRNA increased but not significantly after LPS stimulation. CONCLUSION： JHD can inhibit cytokine secretion pathway induced by LPS in rat liver, which is probably associated with its regulation on CD68, p-IκB and endotoxin receptor TLR2.

Structural Shifts of Gut Flora in Rat Acute Alcoholic Liver Injury and Jianpi Huoxue Decoction's(健脾活血汤)Effect Displayed by ERIC-PCR Fingerprint

Objective： To study the structural shifts of gut flora in rats with acute alcoholic liver injury （AALI）, and the effect of Jianpi Huoxue Decoction （健脾活血汤, JPHXD） on the gut flora. Methods： Thirty-six Sprague- Dawley rats were randomly allocated to the control, AALI and JPHXD groups equally. The rats in the control group were given water and those in AALI and JPHXD groups were given ethanol by intragastric gavage for 5 days, while rats in the JPHXD group were administered JPHXD simultaneously. The blood and liver tissue were collected at the end of the experiment. The activities of serum alkaline aminotransferase （ALT）, aspartate aminotransferase （AST）, hepatic γ/-glutamyitranspetidase （γ-GT） and hepatic tdglyceride （TG） levels were determined. Plasma endotoxin level in the portal vein was measured. Pathological changes of liver tissues were determined by hematoxylin and Eosin （HE） staining and oil red O staining. The total DNA of gut flora were extracted from fecal samples by Bead-beating method and determined by ERIC-PCR fingerprint method. The similarity cluster analysis and principal component analysis were performed to analyze the ERIC-PCR fingerprint respectively. Results： In the AALI group, the ratio of liver/body weight, activities of ALT, AST and hepatic γ-GT, amount of hepatic TG were elevated significantly compared with those in the control group （all P〈0.01）. JPHXD decreased the ratio, activities of ALT, AST, γ-GT and TG significantly compared with those in the AALI group （P〈0.05 or P〈0.01）. HE and oil red O staining showed that fat deposited markedly in liver tissue, while JPHXD alleviated pathological changes markedly. Plasma LPS level in rat portal vein in the AALI group increased significantly （P〈0.01）, but it was decreased significantly in the JPHXD group （P〈0.01）. The cluster analysis and principal component analysis of ERIC-PCR fingerprint showed that gut flora in the hALl group changed markedly, and JPHXD could recover gut flora to some extent. Conclusion： The structure of gut flora shifted markedly during acute alcoholic liver injury, JPHXD had prevention effect through the modification of gut flora.

Intervention Effects of Jianpi Liqi Huoxue Decoction (健脾理气活血汤) on Lipid Peroxidative Liver Injury Induced by Alcohol

Objective： To study the intervention effects of Jianpi Liqi Huoxue Decoction （健脾理气活血汤, JLHD） on lipid peroxidative liver injury induced by alcohol. Methods： The rat alcoholic model of liver disease （ALD） induced by Lieber-DeCarli liquid diet was established. Thirty-two male SD rats were randomly divided into 4 groups ： the normal group （ n = 5）, the control group （ n = 9）, the model group （ n = 9） and the JLHD group （ n =9）. From the 4th week after modeling, the rats were given JLHD or distilled water by gastrogavage respectively, and the samples of blood and liver tissues were taken out from the rats for determination by the end of the 8th week. The hepatic pathological changes were observed with HE staining; the liver injury related indices, including activity of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum, γ-glutamyl transpeptidase （γ-GT） activity and triglyceride （TG） content in liver tissues, as well as the lipid peroxidation related indices, including malonaldehyde （MDA） content and nitric oxide synthase （NOS） activity in liver tissue, serum Fe^2＋ level, and the anti-peroxidation capacity related indices, including superoxide dismutase （SOD） activity, glutathion （GSH） content and reactive oxygen species （anti- ROS） activity in liver tissues were determined. Results： （ 1 ） There were obvious figures of fatty degeneration and inflammatory infiltration in liver tissues of the model group. As compared with the control group, in the model group, the activity of ALT and AST, and Fe^2＋ content in serum, γ-GT and NOS activity, TG and MDA content in liver tissues were significantly higher （ P〈0. 01 ）, while the activity of SOD, GSH and anti-ROS in liver tissues were significantly lower （P〈0.01）. （2） The fatty degeneration and inflammatory infiltration of liver tissues in the JLHD group were significantly lessen as compared with those in the model group; and the abnormalities of all the indexes revealed in the model rats were restored to certain extent in the JLHD group, and especially significant were the levels of ALT activity, MDA content and Fe^2＋ , which were nearly normal. Conclusion： JLHD has significant effects against alcoholic liver injury, the acting mechanism of which is likely to be related with its anti-lipid peroxidative effect.