| Huntington's disease (HD) is a fatal neurodegenerative disorder, all cases of which are caused by an expanded polyglutamine tract, encoded by a CAG repeat sequence in the huntingtin (Htt) gene. HD classically manifests with a triad of signs and symptoms that include motor, cognitive and behavioral features. The polyQ expansion causes marked brain atrophy and induces htt to aggregate into protein deposits termed inclusion bodies which we also called mutant huntingtin (mHtt). The mHtt has many effects in cells, which includes abnormalities in cellular proteostasis mechanisms, enter the nucleus and alter gene transcription, lead to the production of abnormal metabolites and markers of oxidative stress. The mHtt has two forms:the aggregated htt and the soluable htt, the later form has higher toxicity. Reducing the mHtt is considered to be an efficient methord to alleviate HD.Lycium barbarum has been regarded as an upper-class Chinese medicine in the Chinese pharmacopoeia and has been used for its anti-aging properties for centuries. The anti-aging function of LBP has many aspects, some of which include neuroprotective effects, for instance against ?-amyloid peptide neurotoxicity. HD shares many pathological features with other neurodegenerative diseases that are caused by protein misfolding, and these pathological features include the age-dependent accumulation of misfolded proteins and the selective degeneration of neuronal cells. Along with its potential effects on HD, LBP has other known effects, such as those related to the p53 pathway, activation of the Nrf2/HO-1 antioxidant pathway, and activation of c-Jun N-terminal kinase (JNK) by phosphorylation. Upregulation of Nrf2 may help defend against damage from reactive oxygen species (ROS), and the activation of phosphoinositide PI3K-AKT pathways protect against apoptosis.The activation of phosphatidylinositol 3-kinase (PI3K) correlates with increased cell survival, and this effect is largely mediated through the activation of a serine/threonine kinase AKT (also known as PKB). In previous studies, phos-phorylation of AKT (Thr-308 and Ser-473) by phosphoinositidedependent protein kinase-1 and serine/threonine kinases, including an integrinlinked kinase, was responsible for AKT activation. The PI3K/AKT pathway promotes cellular survival. Recently, active AKT has been reported to phosphorylate and inactivate components of the JNK pathway, including ASK1, MLK3 and MKK4, thereby inducing antiapoptotic effects. AKT deactivation selectively associated with both caspase-independent and -dependent cell death in multiple cellular systems. In the absence of cytotoxic cell stressors, AKT exerts a restraining effect on cytotoxic pro-cesses. Stressors deactivate Akt, terminating this protective effect.In HD, AKT plays an important role in HD. HD alters the abundance and activity of AKT. In a rat model of HD, AKT was downregulated during neuronal dysfunction prior to the observation of any signs of degeneration. Finally, in postmortem brains of patients with HD, AKT is cleaved, and this appears to be the final step that ensures the irreversible deactivation of AKT and abolishes the ability of AKT to promote survival in cells expressing polyQ-Htt. Decreased levels of AKT and/or activated AKT will reduce Htt phosphorylation, thereby enhancing the toxicity of polyQ-Htt.In this research, we observed the ability of LBP on mHtt of expression and mechanism using HD cells and HD transgenetic mice. The results showed that LBP can reduce the mHtt by activating AKT-Ser473. In this study, we used the stable cells line which stable expression the EGFP-20Q or EGFP-160Q in HEK293 cells, we calles the cells HEK293-20Q and HEK293-160Q cells. The mice were divided into 4 groups:a wild-type mouse group that received distilled water (WT), a wild-type mouse group that received LBP (WT+LBP), a TGitive mouse group (TG), and a positive mouse group that received LBP (TG+LBP).LBP reduces the mHtt cell toxicity For detecting the inhibition effect of LBP on mHtt cell toxicity, we first detect the cell viability and caspase-3 activity in HEK293-160Q cell. The viability of HEK293-20Q and HEK293-160Q cells was upregulated, as determined using the CCK8 assay, following treatment with LBP at 0.384,1.92,9.6,48,240,1,200,6,000 or 30,000?g/mL for 24 h. At 9.6?g/mL, LBP significantly enhanced the growth of the 2 types of cells, and even at concentration of 30,000?g/mL, the cell viability in HEK293-160Q cells was not significantly decreased, but significantly decreased in HEK293-20Q cells. LBP increases the number of viable cells at lower doses, whilst reducing their number at higher doses. We also analyzing the cell death by caspase-3 activity assay after HEK293-20Q/160Q cells were treated with LBP for 48 h and found that the caspase-3 activity was significantly reduced in HEK293-160Q cells, but no significantly changed in HEK293-20Q cells.LBP prolongs life span, increases body weigh and improves moter function in HD mice HD mice display progressive phenotypes that resemble those observed in humans with Huntington's disease, such as a shorter life span, motor deficits and weight loss. The results of this study showed that LBP can prolong the lifespan of HD mice, significantly reduce weight loss and promote motor functions. All the above phenotypes confirm that LBP can prolong life span, enhance body weight and improve the performance of motor functions in HD mice. All results above confirmed LBP can alleviate the HD symptoms.LBP reduces mutant huntingtin levels Because LBP could enhance cell viability and reduce the cell death, we investigated whether LBP could affect the mHtt in HEK293-20Q/160Q cells. After 7 d, we detected the Htt levels again and found that the levels of both soluble Htt and aggregated Htt were significantly decreased in HEK293-160Q cells, and these results were also verified using the filter trap assay. However, the LBP treatment did not influence the expression of Htt in HEK293-20Q cells.Because LBP could affect the mutant Htt in HEK293-20Q/160Q cells and could prolongs life span, enhance body weight and improves motor performance in TG mice, we investigated the changes in mHtt in TG mice. Western blotting analyses were performed to quantify the expression of mHtt in the TG and TG+LBP groups. These analyses showed that LBP significantly reduced the two types of mHtt in the cortex, hippocampus and striatum. In addition, the immune-histochemistry results also confirmed that mHtt was decreased in the brain. To detect changes in mHtt at the mRNA level, we performed reverse-transcription PCR; we observed no changes, indicating that the reduction in mHtt occurred after transcription.LBP reduces the mHtt cell toxity by increasing the AKT activity After 24 h of treatment with AKT inhibitor LY294002 and/or LBP, the phosphorylation of Ser473 in AKT showed a significant increase in HEK293-160Q cells, indicating AKT activation. After LY294002 and LBP treatment, p-AKT-Ser473 was also increased compared with the LY294002-treated group in both HEK293-20Q and -160Q cells, but no changes were observed between the control and the LBP group in HEK293-20Q cells. We also found the AKT activity significantly decreased in HEK293-160Q cells by comparing to HEK293-20Q cells. We also detected the phosphorylation on Ser9 of GSK3? and the total levels of GSK3?,which was first identified as a substrate of AKT in 1995. Phosphorylation of Ser9 in GSK3? did not significantly change after treatment with LY294002, LBP or LBP+LY294002 in either HEK293-20Q or HEK293-160Q cells.In HD, AKT plays an important role in protecting neurons from death; phosphorylation of Ser473 in AKT is reduced in HD. These results were confirmed in the TG mice in such brain regions as the cortex, hippocampus and striatum. In our results, after LBP treatment, AKT was activated by phosphorylation of Ser473 and was significantly increased in the cortex, hippocampus and striatum in TG+LBP mice. However, no differences were observed when comparing the WT and WT+LBP groups, a result that was also consistent with results obtained with HEK293-20Q cells. The levels of phosphorylated Ser9 in GSK3P showed no significant changes among the groups. After 7 d, we detected the Htt levels again and found that the levels of both soluble and aggregated mHtt significantly decreased by LBP treatment and that the levels of both the soluble and aggregated mHtt level significantly increased after LY294002 treatment. These results were also verified using the filter trap assay. However, LBP and/or LY294002 treatment did not influence the expression of Htt in HEK293-20Q cells.After LY294002 and/or LBP treatment, cell survival assays showed that cell viability was significantly increased in the LY294002+LBP groups compared to LY294002 groups both in HEK293-20Q and 160Q cells; after LY294002 treated, the cell viability was significantly decreased in HEK293-20Q/160Q cells. Caspase-3 activity assay was also detected for analyzing cell death, and its result was significantly decreased in the LY294002+LBP groups compared to LY294002 treatment in both HEK293-20Q and HEK293-160Q cells.Conclusions:LBP can resuce the cell toxicity of mHtt by increasing the AKT activity. LBP can reduce the expression of mHtt in HEK293-160Q cells and HD transgenetic mice brains, increase the cell viability, reduce the caspase-3 activity, prolong the life span of HD transgenetic mice, increase the body weight and improve the moter function. |
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