Clinical Efficacy of Gandou Fumu Granules on Wilson Disease with Liver-kidney Deficiency and Phlegm-blood Stasis / 中国实验方剂学杂志

ObjectiveTo observe the clinical efficacy of Gandou Fumu granules （GDFM） in the treatment of Wilson disease （WD） with liver-kidney deficiency and phlegm-blood stasis. MethodNinety WD patients in The First Affiliated Hospital of Anhui University of Chinese Medicine were randomly divided into a control group （45 cases） and a treatment group （45 cases）. All patients were treated with sodium 2，3-dimercaptopropane-1-sulfonate （DMPS）， while those in the treatment group received additional GDFM. All patients were treated for four courses （32 days）. The traditional Chinese medicine （TCM） syndrome scores，clinical effective rate，24 h urinary copper，ceruloplasmin （CER），tumor necrosis factor-α（TNF-α），interleukin-1β（IL-1β），interleukin-6 （IL-6），superoxide dismutase （SOD），glutathione peroxidase （GSH-Px） and malondialdehyde （MDA） levels of the two groups before and after treatment were observed. ResultAfter treatment， the TCM syndrome scores of the two groups decreased （P<0.01），and the score of TCM syndrome in the treatment group was lower than that of the control group （P<0.01）. The total effective rate of the treatment group was 82.22% （37/45）， higher than 57.78% （26/45） of the control group （χ2=6.402，P<0.05）. There was no significant difference in CER before and after treatment in both groups. The post-treatment 24 hour urinary copper increased （P<0.01）， which was higher in the treatment group than that in the control group （P<0.05）. The TNF-α，IL-1β， and IL-6 levels were significantly reduced in both groups after treatment（P<0.01），and the above indicators in the treatment group were significantly lower than those in the control group （P<0.01）. After treatment，the SOD level increased and the MDA level decreased in the control group （P<0.01）， while no significant difference in GSH-Px level was observed. The SOD and GSH-Px levels increased and the MDA level decreased in the treatment group （P<0.01）. After treatment， SOD and GSH-Px levels of the treatment group were higher than those in the control group， while the MDA level was lower than that in the control group（P<0.05，P<0.01）. ConclusionGDFM can improve the TCM syndrome score and clinical efficacy，enhance the copper removing effect，and inhibit the inflammatory response and antioxidative stress in the treatment of WD with liver and kidney deficiency and phlegm-blood stasis.

Intervention of Gandou Fumu Decoction on Liver Fibrosis in Mice with Wilson's Disease Through JNK Signaling Pathway / 中国实验方剂学杂志

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.

Effect of Gandou Decoction on Mitophagy in Toxic Milk （TX） Mouse Model of Wilson Disease Based on Pink1/Parkin Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Gandou decoction （GDD） on the mitophagy of hippocampal neurons in toxic milk （TX） mouse model of Wilson disease and explore the protective mechanism of GDD against neuron injury through the PTEN induced kinase 1 （Pink1） /E3 ubiquitin ligase （Parkin） pathway. MethodSixty mice were randomly divided into a blank group， a model group， a penicillamine group （0.09 g·kg-1）， and low- （5.5 g·kg-1）， medium- （11 g·kg-1）， and high-dose （22 g·kg-1） GDD groups， and treated correspondingly by gavage for 8 weeks. Morris water maze， traction test， and pole test were used for the evaluation of animal behaviors. Hematoxylin-eosin （HE） staining and transmission electron microscopy were used to observe cell apoptosis， ultrastructure， autophagy， and mitochondrial structure. The levels of superoxide dismutase （SOD）， reactive oxygen species （ROS）， and malondialdehyde （MDA） were detected by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of Pink1， Parkin， autophagy-associated protein Beclin-1， microtubule-associated protein 1 light chain 3Ⅱ （LC3Ⅱ）， and p62. Western blot was conducted to detect the protein expression of Pink1， Parkin， Beclin-1， LC3Ⅱ/Ⅰ， and p62. ResultCompared with the blank group， the model group showed prolonged escape latency， decreased times of platform crossing， lower score in the traction test， and longer pole climbing time （P<0.01）. Compared with the model group， the medium- and high-dose GDD groups and the penicillamine group showed shortened escape latencies， increased times of platform crossing， higher scores in the traction test， and shortened pole climbing time （P<0.01）. Compared with the blank group， the model group displayed severely damaged neurons and increased autophagosomes. Compared with the model group， the medium- and high-dose GDD groups and the penicillamine group showed improved neuron damage and reduced autophagosomes. The levels of ROS and MDA were higher and SOD was lower in the model group than those in the blank group （P<0.01）， while the levels of the above indicators were reversed by GDD intervention as compared with the model group （P<0.01）. Compared with the blank group， the model group exhibited up-regulated mRNA and protein expression of Pink1， Parkin， LC3Ⅱ， and Beclin-1 and down-regulated p62 （P<0.05）. Compared with the model group， the medium- and high-dose GDD groups showed reduced mRNA and protein expression of Pink1， Parkin， LC3Ⅱ， and Beclin-1 and increased p62 （P<0.05， P<0.01）. ConclusionGDD can significantly inhibit the excessive mitophagy in neurons of TX mice and protect neurons from damage. The mechanism may be related to the regulation of the Pink1/Parkin pathway.

Expert consensus on clinical application of GBE50 Dispersible Tablets for ischemic cardiovascular and cerebrovascular diseases / 中国中药杂志

Ginkgo biloba Extract( GBE50) Dispersible Tablets is a new standardized prescription,which is widely used in the treatment of ischemic cardiovascular and cerebrovascular diseases. However,there are still many problems in its clinical application.Rational and safe use of GBE50 Dispersible Tablets is pivotal to the medication safety and clinical prognosis of patients. This consensus has been jointly formulated by clinical experts of traditional Chinese medicine and western medicine in cardiovascular and cerebrovascular diseases and followed the Manual for the Clinical Experts Consensus of Chinese Patent Medicine published by the China Association of Chinese Medicine. The present study identified clinical problems based on clinical investigation,searched the research papers according to PICO clinical problems,carried out evidence evaluation,classification,and recommendation by GRADE system,and reached the expert consensus with nominal group technique. The consensus combines evidence with expert experience. Sufficient evidence of clinical problems corresponds to " recommendations",while insufficient evidence to " suggestions". Safety issues of GBE50 Dispersible Tablets,such as indications,usage and dosage,and medication for special populations,are defined to improve clinical efficacy,promote rational medication,and reduce drug risks. This consensus needs to be revised based on emerging clinical issues and evidencebased updates in practical applications in the future.

Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl2 / 中国实验方剂学杂志

Objective:To explore the effect of Gandou Fumu decoction （GDFMD） on the oxidative damage of HepG2 cells induced by CuCl₂based on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. Method:CuCl₂ （200 μmol·L^-1） 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 + CuCl₂ + normal rat serum）， a GDFMD group （HepG2 cells + CuCl₂ + 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 CuCl₂， with the underlying mechanism related to the activation of PI3K/Akt/mTOR signalling pathway.