Pre-heating stress associated with acute oral leucine supplementation effects in rat gastrocnemius muscle: Implications for protein synthesis signaling pathways.

Skeletal muscle is a highly plastic tissue. The role of heat shock protein 72 (Hsp72) in heat stress-induced skeletal muscle hypertrophy has been well demonstrated; however, the precise mechanisms remain unclear. Essential amino acids, such as leucine, mainly mediate muscle protein synthesis. We investigated the effects of pre-heating and increased Hsp72 expression on the mechanistic target of rapamycin (mTOR) signaling and protein synthesis following leucine administration in rat gastrocnemius muscle. To ensure increased Hsp72 expression in both the red and white portions of the muscle, one leg of male Wistar rats (10-week-old, n = 23) was heat-stressed in 43 °C water for 30 min twice at a 48-h-interval (heat-stressed leg, HS leg). The contralateral leg served as a non-heated internal control (CT leg). After the recovery period (48 h), rats were divided into the pre-administration or oral leucine administration groups. We harvested the gastrocnemius muscle (red and white parts) prior to administration and 30 and 90 min after leucine treatment (n = 7-8 per group) and intramuscular signaling responses to leucine ingestion were determined using western blotting. Heat stress significantly upregulated the expression of Hsp72 and was not altered by leucine administration. Although the phosphorylation levels of mTOR/S6K1 and ERK were similar regardless of heating, 4E-BP1 was less phosphorylated in the HS legs than the CT legs after leucine administration in the red portion of the muscles (P < 0.05). Moreover, c-Myc expression differed significantly after leucine administration in both the red and white portions of the muscles. Our findings indicate that following oral leucine administration, pre-heating partially blunted the muscle protein synthesis signaling response in the rat gastrocnemius muscle.

Diabetes mellitus aggravates humoral immune response in myasthenia gravis by promoting differentiation and activation of circulating Tfh cells.

Myasthenia gravis (MG) is a T-cell-dependent, antibody-mediated autoimmune disease. Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia and emerging evidence indicates its profound impacts on the immune homeostasis. Previous studies and our data showed DM might serve as an independent risk factor of MG, yet the underlying immune and molecular mechanisms remain to be addressed. Our study observed that circulating Tfh (cTfh) cells were increased in MG patients with DM and expressed a high level of ICOS. Besides, positive correlations between activated cTfh cells and plasmablasts were documented. Further studies demonstrated hyperglycemia promoted the differentiation and activation of Tfh cells which, in turn, caused abnormal plasmablasts differentiation and antibody secretion through the mTOR signaling pathway. These results indicated DM might aggravate the aberrant humoral immunity in MG patients by augmenting Tfh cells differentiation and function and tight glycemic control might be beneficial for MG patients with DM.

Effect of Shugan Jianpi Jiedu Prescription Medicated Serum on Proliferation， Migration and Invasion of Triple-negative Breast Cancer MDA-MB-231 Cells Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

Objective:To study the efficacy and mechanism of Shugan Jianpi Jiedu prescription （SJJ） in the treatment of triple-negative breast cancer through <italic>in vitro</italic> cell experiments. Method:The following groups were set up in this study： a normal serum group，a pirarubicin group，and low-，medium-， and high-dose SJJ-medicated serum groups. Twenty SD rats were randomly divided into four groups and administered with SJJ solution （16.8，8.2，4.05 g·kg^-1） and normal saline （equal volume） according to the body surface area to prepare serum. MDA-MB-231 cells were treated separately. The proliferation， migration and invasion of MDA-MB-231 cells were detected by the cell counting kit-8（CCK-8），wound healing assay and transwell cell invasion assay. The phosphoinositide 3-kinase （PI3K），protein kinase B （Akt）， and mechanistic target of rapamycin （mTOR） protein expression levels in MDA-MB-231 cells were tested by the Western blot. Result:The cell proliferation in the three different doses of medicated serum groups and the pirarubicin positive control group was significantly inhibited as compared with that in the normal serum group（<italic>P</italic><0.01），and there was no statistical difference for this between the medium/high dose medicated serum group and the pirarubicin positive control group.The wound healing in the SJJ-medicated serum groups and the pirarubicin group was slowed down as compared with that in the normal serum group （<italic>P</italic><0.01），and the effect in the SJJ-medicated serum groups was weaker than that in the pirarubicin group （<italic>P</italic><0.05，<italic>P</italic><0.01）. The number of cells invading the lower transwell chamber was decreased as compared with that in the normal serum group （<italic>P</italic><0.01），and there was no statistical difference between the medium-/high-dose SJJ-medicated serum groups and the pirarubicin group. Western blot results showed that 48 h after treatment，the PI3K，Akt， and mTOR expression levels in the cells of SJJ-medicated serum groups and the pirarubicin group were lower than those of the normal serum group（<italic>P</italic><0.01）. Conclusion:The SJJ-medicated serum could inhibit the proliferation， migration and invasion of MDA-MB-231 cells presumedly by down-regulating the protein expression levels in the PI3K/Akt/mTOR signaling pathway.

microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1.

microRNA (miR) has been shown to be involved in the treatment of diseases such as osteoarthritis (OA). This study aims to investigate the role of miR-206 in regulating insulin-like growth factor-1 (IGF-1) in chondrocyte autophagy and apoptosis in an OA rat model via the phosphoinositide 3-kinase (P13K)/protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) signaling pathway. Wistar rats were used to establish the OA rat model, followed by the observation of histopathological changes, Mankin score, and the detection of IGF-1-positive expression and tissue apoptosis. The underlying regulatory mechanisms of miR-206 were analyzed in concert with treatment by an miR-206 mimic, an miR-206 inhibitor, or small interfering RNA against IGF-1 in chondrocytes isolated from OA rats. Then, the expression of miR-206, IGF-1, and related factors in the signaling pathway, cell cycle, and apoptosis, as well as inflammatory factors, were determined. Subsequently, chondrocyte proliferation, cell cycle distribution, apoptosis, autophagy, and autolysosome were measured. OA articular cartilage tissue exhibited a higher Mankin score, promoted cell apoptotic rate, increased expression of IGF-1, Beclin1, light chain 3 (LC3), Unc-51-like autophagy activating kinase 1 (ULK1), autophagy-related 5 (Atg5), caspase-3, and Bax, yet exhibited decreased expression of miR-206, P13K, AKT, mTOR, and Bcl-2. Besides, miR-206 downregulated the expression of IGF-1 and activated the P13K/AKT signaling pathway. Moreover, miR-206 overexpression and IGF-1 silencing inhibited the interleukins levels (IL-6, IL-17, and IL-18), cell apoptotic rate, the formation of autolysosome, and cell autophagy while promoting the expression of IL-1ß and cell proliferation. The findings from our study provide a basis for the efficient treatment of OA by investigating the inhibitory effects of miR-206 on autophagy and apoptosis of articular cartilage in OA via activating the IGF-1-mediated PI3K/AKT-mTOR signaling pathway.

Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group.

BACKGROUND: Pediatric paired box 3:forkhead box protein O1 fusion-negative (PF-) rhabdomyosarcoma (RMS) represents a diverse spectrum of tumors with marked differences in histology, myogenic differentiation, and clinical behavior. METHODS: This study sought to evaluate the clinical and mutational spectrum of 24 pediatric PF- human RMS tumors with high levels of myogenic differentiation. Tumors were sequenced with OncoPanel v.2, a panel consisting of the coding regions of 504 genes previously linked to human cancer. RESULTS: Most of the tumors (19 of 24) arose at head/neck or genitourinary sites, and the overall survival rate was 100% with a median follow-up time of 4.6 years (range, 1.4-8.6 years). RAS pathway gene mutations were the most common mutations in PF-, highly differentiated RMS tumors. In addition, Hedgehog (Hh) and mechanistic target of rapamycin (mTOR) gene mutations with evidence for functional relevance (high-impact) were identified in subsets of tumors. The presence of Hh and mTOR pathway gene mutations was mutually exclusive and was associated with high-impact RAS pathway gene mutations in 3 of 4 Hh-mutated tumors and in 1 of 6 mTOR-mutated tumors. CONCLUSIONS: Interestingly, Hh and mTOR gene mutations were previously associated with rhabdomyomas, which are also known to preferentially arise at head/neck and genitourinary sites. Findings from this study further support the idea that PF-, highly differentiated RMS tumors and rhabdomyomas may represent a continuous spectrum of tumors. Cancer 2018;124:1973-81. © 2018 American Cancer Society.