Part 2 Preventive Effects Of Phenylethanol Glycosides From Cistanche Tubulosa On Bovine Serum Albumin-induced Hepatic Fibrosis in Rats
Mar 06, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
NF-κB p65 and collagen I expression after drug intervention in HSC-T6 cells In order to characterize signals of hepatic fibrosis, two key regulatory genes in the liver were determined via RT-PCR assay. The data showed that tHSC-T6 cells from the control group had lower levels of NF- κB and collagen type I mRNA. Conversely, TGF-β1 induced HSC-T6 cells to markedly upregulate NF-κB (P < 0.01) and Col-I (P < 0.01) mRNA, with levels higher than those in the control group. In the presence of different concentrations of CPhGs(phenylethanoid glycoside from cistanche) , the results of NF-κB p65 [P NF-κB = 0.001 for 100 ug/ml, P NF-κB = 0.002 for 75 ug/ml, P NF-κB = 0.007 for 50 ug/ ml, and P NF-κB = 0.012 for 25 ug/ml, respectively] and Col-I [P Col-I = 0.006, P Col-I = 0.009, P Col-I = 0.014, P Col-I = 0.019, respectively] showed reduced expressions of these mRNAs (Fig. 7).
Western blot analysis of collagen I level after drug intervention in HSC-T6 cells Figure 8 shows the collagen I protein expression levels in HSC-T6 cells of the different experimental groups. The collagen I protein expression level was significantly decreased in the various dose groups of CPhGs(phenylethanoid glycoside from cistanche) (100 ug/ml, 75 ug/ml, 50 ug/ml, and 25 ug/ml) compared with the TGF-β1 group
Discussion
CPhGs(phenylethanoid glycoside from cistanche) is a phenylethanoid glycoside isolated and purified from the rhizome of Cistanche, which is used as a traditional Chinese herbal medicine. In recent years, CPhGs had been shown to possess a powerful ability to prevent liver injuries [20]. Therefore, we aimed to investigate whether CPhGs have inhibitory effects on hepatitis fibrosis by BSA-induced hepatic fibrosis in rats. BJRG is commonly used as a therapeutic drug for hepatic fibrosis in China. It is made from turtle shell. Radix Paeonia rubra, Cordyceps Sinensis, Radix isatidis, and etc. have the effects of replenishing Qi and blood, relieving fatigue, softening nodes. In addition, previous studies show that it has the obvious function of blocking early liver fibrosis, inhibiting the proliferation of fat-storing cells, and reducing collagen synthesis [21]. Therefore, BJRG was used as a positive drug in this study.
The pathological changes of hepatic fibrosis in rats induced by BSA injections are similar to those in human portal cirrhosis [22]. CPhGs(phenylethanoid glycoside from cistanche) dose-dependently alleviated the degree of liver fibrosis and inhibited HSC transformation into myofibroblast-like cells, reduced the elevated levels of serum ALT, AST, HA, LN, CIV, TGF-β1 and the liver index, and markedly suppressed expression of collagen I, collagen III and TGF-β1 in liver tissue. The stages of hepatic fibrosis are correlated with the serum levels of HA, LN and IV-C, which as markers may play a role in detecting the degree of hepatic fibrosis [23]. It has been reported that HA is the major resource of the extracellular matrix. IV-C as the essential element of the basement membrane will be synthesized abundantly and deposited heavily in the earlier phases of liver cirrhosis. The serum levels of LN and IV-C are the indexes of the turnover rate of the basement membrane and show the degree of fibrosis in the portal area and sinusoidal capillaries [24]. PC III is a marker in the diagnosis of hepatic fibrosis and early cirrhosis, but its sensitivity and specificity are not high, and there is no significant difference between the various stages of fibrosis in many references [24, 25].
This study obtained a similar result. In addition, the H&E and Masson’s trichrome-stained section observations exhibit normal liver tissues with distinct hepatic lobules and hepatic sinusoids. The liver tissue structure in the model group was disordered, and the liver tissue and hepatic sinusoid were replaced by a large amount of connective tissue. However, significant improvement was observed in the treatment groups compared with the model group. Importantly, collagen type I and collagen type III expression play essential roles in the development of hepatic fibrosis, the blocking of which can prevent and treat hepatic fibrosis. Therefore, the generation and deposition in the liver tissue of collagen type I and collagen type III could serve as an important determinant of anti-hepatic fibrosis efficacy. TGF-β1 is also an important profibrogenic cytokine in liver injury and it is biologically active with multiple pharmacological actions [26]. A balance among these actions is required to maintain tissue homeostasis. The aberrant expression of TGF- β1 is involved in the pathogenesis of liver diseases [27, 28]. It is known that TGF-β1 is a crucial cytokine that is involved in the early stages of liver fibrosis.
Oxidative stress triggers TGF-β1, resulting in the latter stimulating ECM production and deposition [29]. Therefore, one of the effective strategies to produce anti-hepatic fibrosis drug is to identify anti-TGF-β1 agents. Immunohistochemical analysis showed that the expressions of collagen type I, collagen type III and TGF-β1 could detect the pathological process of hepatic fibrosis. The expressions of the collagen type I, collagen type III and TGF-β1 in the treatment groups are decreased, which were significantly lower in the high dose CPhGs(phenylethanoid glycoside from cistanche) treatment group in particular, and suggested that CPhGs is an effective collagen type I, collagen type III and TGF-β1 inhibitor. Presumably, CPhGs can improve collagenase activity, maintaining the dynamic equilibrium of ECM synthesis and degradation, thus delaying and preventing the formation of liver fibrosis. CPhGs(phenylethanoid glycoside from cistanche) not only could ameliorate BSA-induced hepatic fibrosis in rats but also might be associated with inhibiting the activation of HSC in vitro. HSC activation is thought to represent the crucial step of fibrogenesis. In this study, the results illustrated that administration of CPhGs from 25 to 100 ug/ml remarkably attenuated the decreased NF-κB p65, collagen I mRNA expression, and collagen I protein expression in HSC. NF-κB plays an important role in modulating the immune response to infection or stimuli [30]. The buildup of NF-κB in liver cells can result in the recruitment of inflammatory cytokines/mediators, thus inducing fibrosis development [31, 32]. Moreover, collagen is also a sensitive index that reflects the fibrosis level and accounts for about 50 % of the total protein in the fibrous liver [33].
As a result, we postulated that the molecular mechanism against hepato-fibrosis is linked to CPhGs(phenylethanoid glycoside from cistanche)-mediated inactivation of NF-κB expression, in which the benefit contributes to synergistic roles of attenuating immunotoxicity and inflammation stress in BSA-lesioned liver tissue, further correcting dysmetabolism to ameliorate liver functions.
Conclusions
In conclusion, our studies indicate that CPhGs(phenylethanoid glycoside from cistanche) significantly attenuate the extent of hepatic fibrosis induced by BSA in rats. Its mechanism may at least partially be due to the inhibitory effect of CPhGs on the composition of ECM and stimulation of the degradation of ECM, and/ or by direct inhibition of the synthesis of collagen type I, collagen type III and the expression of TGF-β1. Therefore, we expected that CPhGs can be used in health care products or in clinical medications for the prevention of human liver fibrosis. Future studies are required to establish the efficacy of CPhGs as a potent anti-hepatic fibrosis drug.
Abbreviations
CPhGs: Phenylethanol glycosides from cistanche; BSA: Bovine serum albumin; HSC-T6: Hepatic stellate cells; Hyp: Hydroxyproline; BJRG: Compound Biejiarangan tablets; TGF-β1: Transforming growth factor β1; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HA: Hyaluronic acid; LN: Laminin; PC III: Type III procollagen; IV-C: Type IV collagen; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; RT-PCR: Reverse transcriptase-polymerase chain reaction; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Competing interests The authors declare that they have no competing interests. Authors’ contributions TL, JZ, SPY, LM, MT and SLZ conceived and designed the experiments. SPY, TL and JZ analyzed the data. SPY and JZ wrote the manuscript. TL, LM and JZ reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgment This research was supported by the National Natural Science Foundation of China (81260624). The authors would like to express their sincere thanks to Professor Tao Liu for his suggested improvements for the writing of this paper. Author details 1 Department of Toxicology, School of Public Health, Xinjiang Medical University, No. 393 Xinyi Road, Urumqi 830011Xinjiang Uyghur Autonomous
Region, China. 2 Key Laboratory for Uighur Medicine, Institute of Materia Medica of Xinjiang, Urumqi 830004, China. 3 No. 140 Xinhua South Road, Tianshan District, Urumqi 830000Xinjiang Uyghur Autonomous Region, China. Received: 6 August 2015 Accepted: 24 November 2015
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