Electroacupuncture Promotes Functional Recovery after Facial Nerve Injury in Rats by Regulating Autophagy via GDNF and PI3K/mTOR Signaling Pathway.

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS: EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.

Human mesenchymal stem cells exhibit altered mitochondrial dynamics and poor survival in high glucose microenvironment.

BACKGROUND: Mesenchymal stem cells (MSCs) are a type of stem cells that possess relevant regenerative abilities and can be used to treat many chronic diseases. Diabetes mellitus (DM) is a frequently diagnosed chronic disease characterized by hyperglycemia which initiates many multisystem complications in the long-run. DM patients can benefit from MSCs transplantation to curb down the pathological consequences associated with hyperglycemia persistence and restore the function of damaged tissues. MSCs therapeutic outcomes are found to last for short period of time and ultimately these regenerative cells are eradicated and died in DM disease model. AIM: To investigate the impact of high glucose or hyperglycemia on the cellular and molecular characteristics of MSCs. METHODS: Human adipose tissue-derived MSCs (hAD-MSCs) were seeded in low (5.6 mmol/L of glucose) and high glucose (25 mmol/L of glucose) for 7 d. Cytotoxicity, viability, mitochondrial dynamics, and apoptosis were deplored using specific kits. Western blotting was performed to measure the protein expression of phosphatidylinositol 3-kinase (PI3K), TSC1, and mammalian target of rapamycin (mTOR) in these cells. RESULTS: hAD-MSCs cultured in high glucose for 7 d demonstrated marked decrease in their viability, as shown by a significant increase in lactate dehydrogenase (P < 0.01) and a significant decrease in Trypan blue (P < 0.05) in these cells compared to low glucose control. Mitochondrial membrane potential, indicated by tetramethylrhodamine ethyl ester (TMRE) fluorescence intensity, and nicotinamide adenine dinucleotide (NAD+)/NADH ratio were significantly dropped (P < 0.05 for TMRE and P < 0.01 for NAD+/NADH) in high glucose exposed hAD-MSCs, indicating disturbed mitochondrial function. PI3K protein expression significantly decreased in high glucose culture MSCs (P < 0.05 compared to low glucose) and it was coupled with significant upregulation in TSC1 (P < 0.05) and downregulation in mTOR protein expression (P < 0.05). Mitochondrial complexes I, IV, and V were downregulated profoundly in high glucose (P < 0.05 compared to low glucose). Apoptosis was induced as a result of mitochondrial impairment and explained the poor survival of MSCs in high glucose. CONCLUSION: High glucose impaired the mitochondrial dynamics and regulatory proteins in hAD-MSCs ensuing their poor survival and high apoptosis rate in hyperglycemic microenvironment.

Autophagy inhibition enhances colorectal cancer apoptosis induced by dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.

Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway performs a central role in tumorigenesis and is constitutively activated in many malignancies. As a novel dual PI3K/mTOR inhibitor currently undergoing evaluation in a phase I/II clinical trial, NVP-BEZ235 indicates a significant antitumor efficacy in diverse solid tumors, including colorectal cancer (CRC). Autophagy is a catabolic process that maintains cellular homeostasis and reduces diverse stresses through lysosomal recycling of the unnecessary and damaged cell components. This process is also observed to antagonize the antitumor efficacy of PI3K/mTOR inhibitor agents such as NVP-BEZ235, via apoptosis inhibition. In the present study, we investigated anti-proliferative and apoptosis-inducing ability of NVP-BEZ235 in SW480 cells and the crosstalk between autophagy and apoptosis in SW480 cells treated with NVP-BEZ235 in combination with an autophagy inhibitor. The results revealed that, NVP-BEZ235 effectively inhibit the growth of SW480 cells by targeting the PI3K/mTOR signaling pathway and induced apoptosis. The inhibition of autophagy with 3-methyladenine or chloroquine inhibitors in combination with NVP-BEZ235 in SW480 cells enhanced the apoptotic rate as componets to NVP-BEZ235 alone. In conclusion, the findings provide a rationale for chemotherapy targeting the PI3K/mTOR signaling pathway presenting a potential therapeutic strategy to enhance the efficacy of dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with an autophagy inhibitor in CRC treatment and treatment of other tumors.