ABSTRACT
ObjectiveTo identify the protective effect and possible mechanism of Gandou Fumu decoction (GDFMD) on liver fibrosis in mice with Wilson's disease. MethodA total of 50 homozygous TXJ mice were randomly divided into five groups, with 10 mice in each group. Ten wild-type mice were selected as a normal group. The GDFMD high, medium, and low-dose groups were given 13.92, 6.96, 3.48 g·kg-1 of GDFMD, respectively. The penicillamine group were given 0.1 g·kg-1 of penicillamine. The model group and the normal group were given the same volume of 0.9% sodium chloride solution once a day for 4 consecutive weeks. The enzyme-linked immunosorbent assay (ELISA) method was performed to detect serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Corresponding kits were used to detect the mitochondrial adenine triphosphate (ATP) content and Na+-K+-ATPase activity in liver tissues. Hematoxylin-eosin (HE) and Masson staining were used to observe the pathological morphology of liver tissue, and transmission electron microscope was used to observe ultrastructural changes of liver tissues in mice. Western blot was used to detect the c-Jun N-terminal kinase, the phosphorylated protein, and the expressions of Caspase-3, B cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) in c-Jun N-terminal kinase (JNK) signaling pathway. ResultCompared with the normal group, MDA content increased and SOD activity decreased in the model group (P<0.05). Compared with the model group, SOD activities in the GDFMD high-, medium-, and low-dose groups and the penicillamine group significantly increased (P<0.01), and MDA content significantly decreased (P<0.05, P<0.01). Compared with the normal group, ATP content and Na+-K+-ATPase activity significantly decrease in the model group (P<0.05). Compared with the model group, ATP content and Na+-K+-ATPase activity in the GDFMD medium and high-dose groups and the penicillamine group significantly increased (P<0.05, P<0.01). The results of the pathological morphology of liver tissue showed that a large number of liver cells degeneration and necrosis, inflammatory cell infiltration, unclear liver lobule structure, and collagen fiber deposition were observed in the model group. Transmission electron microscopy showed that the number of mitochondria in liver tissues significantly reduced, the mitochondria were locally damaged, and the cristae of mitochondria were broken even disappear in the model group. The pathological morphology of liver tissue and mitochondrial structure recovered to varying degrees after medicinal intervention. The results of Western blot suggested that, compared with the normal group, the expression levels of phosphorylation-JNK (p-JNK), p-JNK/JNK, Caspase-3, and Bax in the liver tissues were up-regulated, while the expression of Bcl-2 was down-regulated in the model group (P<0.05). The expression levels of p-JNK, p-JNK/JNK, Caspase-3 and Bax were down-regulated and the expression of Bcl-2 was up-regulated in the GDFMD high and medium-dose groups and the penicillamine group (P<0.01). ConclusionGDFMD can alleviate oxidative stress damage and recover mitochondrial function of TXJ mice with liver fibrosis. The mechanism of GDFMD may be related to regulating the JNK signaling pathway and downstream factors and inhibiting cell apoptosis.
ABSTRACT
Hepatolenticular degeneration(HLD),also known as Wilson disease (WD), is a genetic disorder characterized by copper metabolism disorder caused by ATP7B gene mutation. Specifically, due to the ceruloplasmin synthesis disorder induced by gene mutation,copper cannot be excreted through bile,which results in pathological deposition of copper in various organs and damage to organs such as the brain and the liver. The incidence of WD in Chinese is significantly higher than that in the world. Copper chelating agents, such as D-penicillamine and dimercaptosuccinic acid, are used as the main therapeutic agents in western medicine. However, many clinical adverse events limit the application of these drugs. Traditional Chinese medicine (TCM) has its characteristics in the treatment of WD. As confirmed by long-term research on TCM clinical diagnosis and treatment,MD has become TCM dominant disease. In spite of many views about the etiology and pathogenesis of WD,a consensus has not been reached so far. Based on the theory of latent pathogen in TCM and the pathological mechanism of excessive deposition of copper ions in the body,this study proposed that latent toxin is the key etiology of WD,and further elaborated that the latent toxin of WD was inherited from parents and occurred in children and adolescents,which was hidden in the liver and the kidney and damaged the brain. The latent toxin, Yang in nature and dispersing in property, is prone to transform into dampness-heat to block Qi movement and produce phlegm leading to stasis. Furthermore, this study determined latent toxin blocking collaterals as the basic pathogenesis of WD and revealed the complex clinical manifestations of latent toxin blocking collaterals such as liver collaterals,brain collaterals,kidney collaterals,spleen collaterals,stomach collaterals,lung collaterals,heart collaterals, and uterus collaterals. Treatment should follow the basic therapeutic principles of resolving pathogens,removing toxins, and dredging collaterals. This study is expected to provide a theoretical basis for syndrome differentiation and treatment of WD in TCM.
ABSTRACT
Objective:To explore the effect of Gandou Fumu decoction (GDFMD) on the oxidative damage of HepG2 cells induced by CuCl<sub>2 </sub>based on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. Method:CuCl<sub>2</sub> (200 μmol·L<sup>-1</sup>) was used to induce a copper-loaded HepG2 cell model. HepG2 cells were divided into a blank group (HepG2 cells + blank rat serum), a model group (HepG2 cells + CuCl<sub>2</sub> + normal rat serum), a GDFMD group (HepG2 cells + CuCl<sub>2</sub> + GDFMD-medicated rat serum), an inhibitor group (HepG2 cells + NVP-BEZ235 + normal rat serum), and a GDFMD + NVP-BEZ235 group (HepG2 cells + NVP-BEZ235 + GDFMD-medicated rat serum). ELISA method was used to determine superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content. The expression of microtubule-associated protein 1 light chain 3 (LC3) was detected by immunofluorescence. Phospho-PI3K/PI3K (p-PI3K/PI3K), p-Akt/Akt, p-mTOR/mTOR, Beclin-1, LC3Ⅱ/LC3Ⅰ, and p62/Actin were determined by Western blot. PI3K, Akt, mTOR, Beclin-1, LC3Ⅰ, LC3Ⅱ, p62 mRNA expression was measured by real-time polymerase chain reaction (PCR). Result:Compared with the blank group, the model group displayed decreased activities of SOD and GSH-Px and increased content of MDA (<italic>P</italic><0.01). Compared with the model group, the GDFMD group showed elevated activities of SOD and GSH-Px and reduced content of MDA (<italic>P</italic><0.05, <italic>P</italic><0.01), while the inhibitor group exhibited weakened GSH-Px activity and up-regulated content of MDA (<italic>P</italic><0.05). Compared with the blank group, the model group showed diminished expression of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, and p62, and increased expression of Beclin-1 and LC3Ⅱ/LC3Ⅰ (<italic>P</italic><0.01). The expression of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, and p62 was elevated, and the expression of Beclin-1 and LC3Ⅱ/LC3Ⅰ declined in the GDFMD group (<italic>P</italic><0.05,<italic> P<</italic>0.01), while the p-PI3K/PI3K and p-mTOR/mTOR expression was down-regulated and the Beclin-1 and LC3Ⅱ/LC3 I expression was increased in the inhibitor group (<italic>P</italic><0.05, <italic>P<</italic>0.01) as compared with those in the model group. Compared with the GDFMD group, the GDFMD + NVP-BEZ235 group showed down-regulated expression of p-Akt/Akt and p-mTOR/mTOR and up-regulated expression of Beclin-1 and LC3Ⅱ/LC3Ⅰ(<italic>P</italic><0.05, <italic>P</italic><0.01). The expression of LC3Ⅱ protein in the model group was increased (<italic>P</italic><0.01) as compared with that in the blank group. The expression of LC3Ⅱ protein was lower in the GDFMD group than in the model group, and higher in the GDFMD + NVP-BEZ235 group than in the GDFMD group. No significant difference in the expression of PI3K, Akt, and mTOR mRNA was observed among the groups. Compared with the blank group, the model group displayed lowered expression of p62 mRNA, and elevated expression of Beclin-1, LC3Ⅰ, and LC3Ⅱ mRNA (<italic>P</italic><0.01). Compared with the model group, the GDFMD group exhibited increased expression of p62 mRNA, and declining expression of Beclin-1, LC3Ⅰ, and LC3Ⅱ mRNA (<italic>P</italic><0.01), while the inhibitor group showed increased expression of Beclin-1 mRNA (<italic>P</italic><0.05). The expression of Beclin-1 and LC3Ⅱ mRNA in the GDFMD + NVP-BEZ235 group was elevated (<italic>P</italic><0.01) as compared with that in the GDFMD group. Conclusion:GDFMD may inhibit the excessive autophagy and alleviate the oxidative damage of HepG2 cells induced by CuCl<sub>2</sub>, with the underlying mechanism related to the activation of PI3K/Akt/mTOR signalling pathway.
ABSTRACT
BACKGROUND: The treatment of spinal cord injury is mainly to improve the inflammation microenvironment of the injury site, as well as the regeneration and repair of neural function. Mesenchymal stem cells have the characteristics of easy proliferation in vitro, multi-directional differentiation, and suppression of secondary inflammation and immunomodulation, which makes the transplantation of mesenchymal stem cells for various refractory tissue injury diseases including spinal cord injury become a potential cell therapy. OBJECTIVE: To summarize the preclinical and clinical research of mesenchymal stem cells in the treatment of spinal cord injury in recent years, and put forward the problems and development directions of mesenchymal stem cells in experimental research. METHODS: We searched the articles published in Wanfang databases, CNKI and PubMed databases from 2000 to 2020. The key words were “mesenchymal stem cells, spinal cord injury, cell transplantation, immunomodulation, combination therapy, biomaterials” in Chinese and English, respectively. RESULTS AND CONCLUSION: (1) Mesenchymal stem cells from different sources play the role of anti-inflammatory, inducing axon and neuron regeneration in animal models and clinical experiments, and effectively improve the neural function of the damaged area. (2) The exosomes derived from mesenchymal stem cells show the effects of immunomodulation and angiogenesis in the disease model of spinal cord injury. (3) In order to maximize the potential of mesenchymal stem cells, exploring cell pretreatment, combined with new drugs or biological materials is the research direction of mesenchymal stem cells in the treatment of spinal cord injury.