Dihydromyricetin resists inflammation-induced muscle atrophy via ryanodine receptor-CaMKK-AMPK signal pathway.

Skeletal muscle plays a pivotal role in the maintenance of physical and metabolic health. Skeletal muscle atrophy usually results in physical disability, inferior quality of life and higher health care costs. The higher incidence of muscle atrophy in obese and ageing groups is due to increased levels of inflammatory factors during obesity and ageing. Dihydromyricetin, as a bioactive polyphenol, has been used for anti-inflammatory, anti-tumour and improving insulin sensitivity. However, there are no published reports demonstrated the dihydromyricetin effect on inflammation-induced skeletal muscle atrophy. In this study, we first confirmed the role of dihydromyricetin in inflammation-induced skeletal muscle atrophy in vivo and in vitro. Then, we demonstrated that dihydromyricetin resisted inflammation-induced skeletal muscle atrophy by activating Ca2+ -CaMKK-AMPK through signal pathway blockers, Ca2+ probes and immunofluorescence. Finally, we clarified that dihydromyricetin activated Ca2+ -CaMKK-AMPK signalling pathway through interaction with the ryanodine receptor, its target protein, by drug affinity responsive target stability (DARTS). Our results not only demonstrated that dihydromyricetin resisted inflammation-induced muscle atrophy via the ryanodine receptor-CaMKK-AMPK signal pathway but also discovered that the target protein of dihydromyricetin is the ryanodine receptor. Our results provided experimental data for the development of dihydromyricetin as a functional food and new therapeutic strategies for treating or preventing skeletal muscle atrophy.

Radiographic Osteoarthritis Prevalence Over Ten Years After Anterior Cruciate Ligament Reconstruction.

The purpose of this study was to conduct an up-to-date systematic review and meta-analysis of radiographic knee osteoarthritis (OA) over minimal ten years after ACL reconstruction. The database of Pubmed and the Ovid was adopted. The radiographic knee OA over minimal ten years after ACL reconstruction was systematically reviewed. Both the ipsilateral and contralateral knees were evaluated referring to the tibiofemoral joint (TFJ), the patellofemoral joint (PFJ), and the overall knee OA prevalence. Nineteen studies were included for review, with nine screened for the meta-analysis. The overall knee OA rate ranged from 8.3-79.2%, meanly 51.6% on the ipsilateral side; ranged from 3.6-35.7%, meanly 15.5% on the contralateral side. Compared to the contralateral side, the RR of developing radiographic OA was 3.73 (P<0.01) for the overall knee, 2.88 (P<0.01) for TFJ, and 2.42 (P<0.01) for PFJ. Ipsilaterally, the RR of developing TFJ radiographic OA was 1.15 (P<0.01) compared to that of the PFJ. Over a minimum of 10 years after surgery, more than half the cases developed overall radiographic OA on the ipsilateral knee, which was nearly four times higher than the contralateral side. On the ipsilateral knee, the TFJ was most affected.

Anatomical and Biochemical Traits Related to Blue Leaf Coloration of Selaginella uncinata.

Selaginella uncinata shows particularly rare blue leaves. Previous research has shown that structural interference by the cell wall of adaxial epidermal cells imparts blue coloration in leaves of S. uncinata; the objective of this study was to see whether anthocyanins might additionally contribute to this color, as changes in pH, and conjugation with metals and other flavonoids is also known to result in blue coloration in plants. We compared anatomical and biochemical traits of shade-grown (blue) S. uncinata leaves to high light (red) leaves of the same species and also to a non-blue (green) leaves of a congeneric S. kraussiana. By examining the anatomical structure, we found that the shape of adaxial epidermis of S. uncinata leaves was convex or lens-shaped on the lateral view and irregular circles with smooth embossment on the top view. These features were different from those of the abaxial and adaxial epidermis of S. kraussiana. We suspect that these structures increase the proportion of incident light entering the cell, deepening the leaf color, and therefore may be related to blue leaf color in S. uncinata. By examining biochemical traits, we found little difference in leaf pH value among the leaf types; all leaves contained several metal ions such as Mg, Fe, Mn, and copigments such as flavones. However, because there was no anthocyanin in blue S. uncinata leaves, we concluded that blue coloration in S. uncinata leaves is not caused by the three hypotheses of blue coloration: alkalization of the vacuole pH, metal chelation, or copigmentation with anthocyanins, but it may be related to the shape of the leaf adaxial epidermis.

[Effects of electric pulses on liver cancer cells: apoptosis induction and decrease of mitochondrial transmembrane potential].

In order to investigate the effects of electric pulses on cancer cells, we carried out the experiments with exposing HepG2 and L02 to electric pulses (1 kV/cm, l00 micros, 1 Hz) for different lengths of time (8 s, 15 s, 30 s, 60 s). Annexin V-FITC Kit and Flow cytometry were used to study the apoptosis of treated cells. The results showed that the electric pulses of 1 kV/cm, l00 micros, 1 Hz for 8 s could not induce tumor cells apoptosis. Apoptosis was observed when tumor cells were stimulated for 15 s and longer, and the apoptosis percentage increased with the increase of stimulation time. Furthermore, tumor cells were more sensitive than normal cells in response to electrical pulses. Rhodamine 123 and Laser Scanning Confocal Microscope (LSCM) were used to make a real-time study of mitochondrial transmembrane potential (Deltapsim) when the tumor cells were exposed to electric pulses for 60 s. No significant change of Deltapsim was observed within 30 s stimulation. After that, the Deltapsim increased sharply and declined later, suggesting that the mitochondrial pathway may be one of the apoptosis mechanism induced by electric pulses.

Differences in artificial ligament graft osseointegration of the anterior cruciate ligament in a sheep model: a comparison between interference screw and cortical suspensory fixation.

BACKGROUND: Interference screws are the most common femoral fixation for anterior cruciate ligament (ACL) reconstruction with polyethylene terephthalate (PET) artificial ligaments. However, interference screws have several disadvantages, such as the risk of one tunnel blowout and damage to the graft. Suspensory fixations have the advantages of high tensile strength and promotion of graft bone contact. The purpose of this study was to compare PET artificial ligament graft osseointegration between interference screw fixation (ISF) and cortical suspensory fixation (CSF) for ACL reconstruction. METHODS: Forty sheep underwent ACL reconstruction of the right knee with PET artificial ligament. The graft was fixed with ISF or CSF for femoral fixation. Animals were randomly assigned to the ISF (n=20) or the CSF (n=20) groups. The sheep were sacrificed at 3 or 12 months postoperatively for biomechanical tests, micro computed tomography (micro-CT) scans, and histological assessments. RESULTS: The mean load-to-failure between the CSF group (836±355 N) appeared higher than that of the ISF group (604±277 N) at 3 months, but no significant difference was detected between the groups (P=0.24). At 12 months, there was also no significant difference in load-to-failure between the CSF and ISF groups (1,194±350 vs. 1,097±764 N; P=0.78). According to the micro-CT scan results, the femoral bone tunnel diameter of the ISF group appeared larger than that of the CSF group at 3 months (12±1 vs. 10±1 mm; P=0.02) and similar to that of the CSF group at 12 months (12±1 vs. 11±2 mm; P=0.38). Furthermore, histological results showed that at the graft-tunnel interface of the femoral tunnel aperture, disoriented fibers formed in the ISF group while oriented and dense fibers formed in the CSF group. CONCLUSIONS: ACLR with synthetic ligament by cortical suspension devices with adjustable loops demonstrated a better graft-bone healing capacity at the femoral tunnel aperture compared with that from titanium interference screws over 12 months postoperatively. No significant difference was found in biomechanical strength between the two fixation methods during the early healing stage.

Dihydromyricetin Ameliorates Inflammation-Induced Insulin Resistance via Phospholipase C-CaMKK-AMPK Signal Pathway.

Patients with metabolic syndrome have a higher risk of type II diabetes and cardiovascular disease. The metabolic syndrome has become an urgent public health problem. Insulin resistance is the common pathophysiological basis of metabolic syndrome. The higher incidence of insulin resistance in obese groups is due to increased levels of inflammatory factors during obesity. Therefore, developing a therapeutic strategy for insulin resistance has great significance for the treatment of the metabolic syndrome. Dihydromyricetin, as a bioactive polyphenol, has been used for anti-inflammatory, antitumor, and improving insulin sensitivity. However, the target of DHM and molecular mechanism of DHM for preventing inflammation-induced insulin resistance is still unclear. In this study, we first confirmed the role of dihydromyricetin in inflammation-induced insulin resistance in vivo and in vitro. Then, we demonstrated that dihydromyricetin resisted inflammation-induced insulin resistance by activating Ca2+-CaMKK-AMPK using signal pathway blockers, Ca2+ probes, and immunofluorescence. Finally, we clarified that dihydromyricetin activated Ca2+-CaMKK-AMPK signaling pathway by interacting with the phospholipase C (PLC), its target protein, using drug affinity responsive target stability (DARTS) assay. Our results not only demonstrated that dihydromyricetin resisted inflammation-induced insulin resistance via the PLC-CaMKK-AMPK signal pathway but also discovered that the target protein of dihydromyricetin is the PLC. Our results provided experimental data for the development of dihydromyricetin as a functional food and new therapeutic strategies for treating or preventing PLC.

Retears and Concomitant Functional Impairments After Rotator Cuff Repair: Shoulder Activity as a Risk Factor.

BACKGROUND: Most patients return successfully to shoulder involving sports or activity after rotator cuff repairs. It has not been decided yet whether postoperative participation in shoulder activity adds to the risk of retear. PURPOSE/HYPOTHESIS: The purpose was to verify whether patients who participate in shoulder activities after rotator cuff repair have a higher risk of structural failure than nonactive patients and to investigate the relationship between postoperative shoulder function and tendon integrity in active and nonactive patients. The hypotheses were that (1) active patients have a higher retear rate than nonactive patients and (2) structural failure is associated with worse functional outcomes in active patients. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A cohort study was performed using 145 patients who underwent arthroscopic rotator cuff repair from 2015 to 2017. Functional assessments and magnetic resonance imaging were performed at least 24 months postoperatively. Shoulder activities since 6 months after surgery were rated in 4 categories (sports, job, activities of daily life, and weight of general lifting) as sedentary, light, moderate, or strenuous. The activity level of each patient was defined by the highest rated category. Patients who were involved in light, moderate, and strenuous activity were identified as active for the present study, and the rest were defined as sedentary. The proportion of retears between groups and the functional conditions between retorn and intact tendons were compared. RESULTS: A total of 48 patients were enrolled in the active group, and 97 were enrolled in the sedentary group. The active group demonstrated a significantly higher retear rate than the sedentary group (27.1% vs 11.3%, respectively; P = .016; risk ratio, 2.39 [95% CI, 1.16-4.93]). In the active group, patients with retears showed higher visual analog scale scores for pain, decreased abduction strength, and lower shoulder functional scores (American Shoulder and Elbow Surgeons score, Fudan University Shoulder Score, and Constant-Murley score) than those with healed tendons, whereas in the sedentary group, functional outcomes were generally similar across patients with and without retears. CONCLUSION: Shoulder activity after the early postoperative period was associated with a high risk of retears in patients who underwent rotator cuff repair. A correlation between inhibited function and structural failure was detected but only in active patients, while sedentary patients with retears retained functional improvements similar to those with intact tendons.

Bone-Patellar Tendon-Bone Autografts Versus Hamstring Autografts Using the Same Suspensory Fixations in ACL Reconstruction: A Systematic Review and Meta-analysis.

BACKGROUND: Bone-patellar tendon-bone (BPB) autografts and hamstring tendon (HT) autografts are 2 popular choices for anterior cruciate ligament reconstruction (ACLR). Although existing meta-analyses have explored the clinical outcomes between BPB and HT autografts, none have based their analysis on studies with just femoral suspensory fixation methods. PURPOSE: To evaluate and compare clinical outcomes, particularly graft failure and knee stability, of ACLR with BPB or HT autografts with suspensory femoral fixation. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: A literature search was conducted of studies reporting single-bundle ACLR with BPB autografts and HT autografts with suspensory fixation with a minimum 24-month follow-up. Graft failure rate, knee stability, and clinical outcomes were compared for BPB versus HT autografts. Knee stability was measured with the Lachman test, pivot-shift test, and KT-1000/2000 arthrometer side-to-side difference (SSD). Clinical outcomes were measured with Lysholm scores and the Tegner activity scale, as well as rate of return to preinjury sports. Donor site morbidity among included studies was reviewed. A random-effects model was used for calculations of summary estimates. Subgroup, sensitivity, and trial sequential analyses were conducted. RESULTS: Five studies were included. Graft failure was seen more often in the HT group than the BPB group, and this was statistically significant (P = .03). However, the trial sequential analysis outcome indicated that the included sample size was not large enough to support a solid positive finding. The analysis showed no significant difference in SSD, Lachman test, pivot-shift test, rate of return to sports, Lysholm score, or Tegner score between groups. Subgroup analyses found no significant difference between groups. CONCLUSION: This meta-analysis demonstrated no significant differences in knee stability and knee functional outcomes between BPB and HT autografts with suspensory fixation. More evidence is needed to prove the lower risk of failure with use of BTB autograft with suspensory fixation.

MiR-199b represses porcine muscle satellite cells proliferation by targeting JAG1.

Pig is a useful medical model for humans due to its similarity in size and physiology. Skeletal muscle plays an essential role in body movement. However, the skeletal muscle injuries are common. Skeletal muscle function maintenance largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation plays an essential role in postnatal muscle growth and regeneration. Therefore, understanding the mechanisms associated with muscle satellite cells proliferation is essential for devising the alternative treatments for muscle injury. Previous studies showed JAG1-Notch1 signaling pathway and miRNAs regulate the skeletal muscle development. JAG1-Notch1 signal pathway regulates the transcription of certain types of miRNAs which further affects target gene expression. However, the specific relationship between JAG1-Notch1 signal pathway and miRNAs during muscle development has not been established. We found overexpression of intracellular domain of the Notch1 protein (N1ICD) in porcine muscle satellite cells (PSCs) decreased miR-199b level. We demonstrated that miR-199b inhibits PSCs proliferation using the overexpression and inhibition of miR-199b experiment. We also found JAG1, the miR-199b target gene, promotes PSCs proliferation through activating the Notch1 signal pathway. Furthermore, we demonstrated miR-199b forms a feedback loop with the JAG1-Notch1 signal pathway to maintain the PSCs niche homeostasis. Our results of miRNAs and genes work collaboratively in regulating PSCs proliferation expand our understanding in PSCs proliferation mechanism. Furthermore, this finding indicates miR-199b is a potential therapeutic target for muscle atrophy.

PFN2a Suppresses C2C12 Myogenic Development by Inhibiting Proliferation and Promoting Apoptosis via the p53 Pathway.

Skeletal muscle plays a crucial role in physical activity and in regulating body energy and protein balance. Myoblast proliferation, differentiation, and apoptosis are indispensable processes for myoblast myogenesis. Profilin 2a (PFN2a) is a ubiquitous actin monomer-binding protein and promotes lung cancer growth and metastasis through suppressing the nuclear localization of histone deacetylase 1 (HDAC1). However, how PFN2a regulates myoblast myogenic development is still not clear. We constructed a C2C12 mouse myoblast cell line overexpressing PFN2a. The CRISPR/Cas9 system was used to study the function of PFN2a in C2C12 myogenic development. We find that PFN2a suppresses proliferation and promotes apoptosis and consequentially downregulates C2C12 myogenic development. The suppression of PFN2a also decreases the amount of HDAC1 in the nucleus and increases the protein level of p53 during C2C12 myogenic development. Therefore, we propose that PFN2a suppresses C2C12 myogenic development via the p53 pathway. Si-p53 (siRNA-p53) reverses the PFN2a inhibitory effect on C2C12 proliferation and the PFN2a promotion effect on C2C12 apoptosis, and then attenuates the suppression of PFN2a on myogenic differentiation. Our results expand understanding of PFN2a regulatory mechanisms in myogenic development and suggest potential therapeutic targets for muscle atrophy-related diseases.

MiR-501-3p Forms a Feedback Loop with FOS, MDFI, and MyoD to Regulate C2C12 Myogenesis.

Skeletal muscle plays an essential role in maintaining body energy homeostasis and body flexibility. Loss of muscle mass leads to slower wound healing and recovery from illness, physical disability, poor quality of life, and higher health care costs. So, it is critical for us to understand the mechanism of skeletal muscle myogenic differentiation for maintaining optimal health throughout life. miR-501-3p is a novel muscle-specific miRNA, and its regulation mechanism on myoblast myogenic differentiation is still not clear. We demonstrated that FOS was a direct target gene of miR-501-3p, and MyoD regulated miR-501-3p host gene Clcn5 through bioinformatics prediction. Our previous laboratory experiment found that MDFI overexpression promoted C2C12 myogenic differentiation and MyoD expression. The database also showed there is an FOS binding site in the MDFI promoter region. Therefore, we hypothesize that miR-501-3p formed a feedback loop with FOS, MDFI, and MyoD to regulate myoblast differentiation. To validate our hypothesis, we demonstrated miR-501-3p function in the proliferation and differentiation period of C2C12 cells by transfecting cells with miR-501-3p mimic and inhibitor. Then, we confirmed there is a direct regulatory relationship between miR-501-3p and FOS, MyoD and miR-501-3p, FOS and MDFI through QPCR, dual-luciferase reporter system, and ChIP experiments. Our results not only expand our understanding of the muscle myogenic development mechanism in which miRNA and genes participate in controlling skeletal muscle development, but also provide treatment strategies for skeletal muscle or metabolic-related diseases in the future.