PHD3 mediates denervation skeletal muscle atrophy through Nf-κB signal pathway.

Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are the classical regulator of the hypoxia inducible factor (HIF) signal pathway, many researchers found that PHDs are involved in the muscle fiber type transformation, muscle regeneration, and myocyte differentiation. However, whether PHDs can impact the protein turnover of skeletal muscle is poorly understood. In this study, we constructed denervated muscle atrophy mouse model and found PHD3 was highly expressed in the atrophic muscles and there was a significant correlation between the expression level of PHD3 and skeletal muscle weight which was distinct from PHD1 and PHD2. Then, the similar results were getting from the different weight muscles of normal mice. To further verify the relationship between PHD3 and skeletal muscle protein turnover, we established a PHD3 interference model by injecting PHD3 sgRNA virus into tibialis anterior muscle (TA) muscle of MCK-Cre-cas9 mice and transfecting PHD3 shRNA lentivirus into primary satellite cells. It was found that the Knock-out of PHD3 in vivo led to a significant increase in muscle weight and muscle fiber area (P < .05). Besides, the activity of protein synthesis signal pathway increased significantly, while the protein degradation pathway was inhibited evidently (P < .05). In vitro, the results of 5-ethynyl-2'-deoxyuridine (EdU) and tetramethylrhodamine ethyl ester (TMRE) fluorescence detection showed that PHD3 interference could lead to a decrease in cell proliferation and an increase of cell apoptosis. After the differentiation of satellite cells, the production of puromycin in the interference group was higher than that in the control group, and the content of 3-methylhistidine in the interference group was lower than that in the control group (P < .05) which is consistent with the change of protein turnover signal pathway in the cell. Mechanistically, there is an interaction between PHD3, NF-κB, and IKBα which was detected by immunoprecipitation. With the interfering of PHD3, the expression of the inflammatory signal pathway also significantly decreased (P < .05). These results suggest that PHD3 may affect protein turnover in muscle tissue by mediating inflammatory signal pathway. Finally, we knocked out PHD3 in denervated muscle atrophy mice and LPS-induced myotubes atrophy model. Then, we found that the decrease of PHD3 protein level could alleviate the muscle weight and muscle fiber reduction induced by denervation in mice. Meanwhile, the protein level of the inflammatory signal pathway and the content of 3-methylhistidine in denervated atrophic muscle were also significantly reduced (P < .05). In vitro, PHD3 knock-out could alleviate the decrease of myotube diameter induced by LPS, and the expression of protein synthesis pathway was also significantly increased (P < .05). On the contrary, the expression level of protein degradation and inflammatory signal pathway was significantly decreased (P < .05). Through these series of studies, we found that the increased expression of PHD3 in denervated muscle might be an important regulator in inducing muscle atrophy, and this process is likely to be mediated by the inflammatory NF-κB signal pathway.

Diversity effect of capsaicin on different types of skeletal muscle.

Capsaicin is a major pungent content in green and red peppers which are widely used as spice, and capsaicin may activate different receptors. To determine whether capsaicin has different effects on different types of skeletal muscle, we applied different concentrations (0, 0.01, and 0.02%) of capsaicin in the normal diet and conducted a four-week experiment on Sprague-Dawley rats. The fiber type composition, glucose metabolism enzyme activity, and different signaling molecules' expressions of receptors were detected. Our results suggested that capsaicin reduced the body fat deposition, while promoting the slow muscle-related gene expression and increasing the enzyme activity in the gastrocnemius and soleus muscles. However, fatty acid metabolism was significantly increased only in the soleus muscle. The study of intracellular signaling suggested that the transient receptor potential vanilloid 1 (TRPV1) and cannabinoid receptors in the soleus muscle were more sensitive to capsaicin. In conclusion, the distribution of TRPV1 and cannabinoid receptors differs in different types of muscle, and the different roles of capsaicin in different types of muscle may be related to the different degrees of activation of receptors.

Phytol increases adipocyte number and glucose tolerance through activation of PI3K/Akt signaling pathway in mice fed high-fat and high-fructose diet.

It has been shown that adipose tissue hyperplasia (increased adipocyte number or adipogenesis) has beneficial effects on metabolic health. The aim of the present study was to determine whether phytol could modulate hyperplasia/adipogenesis and glucose homeostasis, and to explore the underlying mechanisms in mice fed high-fat and high fructose diet (HFFD). Our results demonstrated that phytol administration decreased body weight gain and inguinal subcutaneous white adipose tissue (iWAT) weight. However, phytol significantly increased the adipocyte number in iWAT, with the smaller average adipocyte diameter. Meanwhile, OGTT result showed that phytol improved glucose tolerance. In accord, phytol administration markedly increased expression of marker genes associated with adipogenesis (PPARγ and C/EBPα) and glucose uptake (AS160 and GLUT4) and activated PI3K/Akt signaling pathway in mice iWAT. In agreement with the in vivo findings, the in vitro results indicated that 100 µM phytol significantly enhanced 3T3-L1 adipogenesis and glucose uptake, and activated PI3K/Akt signaling pathway. However, phytol-induced enhancement of 3T3-L1 adipognesis and glucose uptake, activation of PI3K/Akt signaling pathway, elevation of marker genes involved in adipogensis and glucose uptake, as well as translocation of GLUT4 from cytoplasm to membrane were abolished by Wortmannin, a specific PI3K/Akt inhibitor. Taken together, phytol increased adipocyte number in iWAT and improved glucose tolerance in mice fed HFFD, which was coincident with the enhanced adipogenesis and glucose uptake in 3T3-L1, and was associated with activation of PI3K/Akt signaling pathway. These data suggested the application of phytol as a potential nutritional agent to combat obesity and type 2 diabetes.

Effect of maternal dietary supplementation with phytosterol esters on muscle development of broiler offspring.

Recently, embryo muscle development, which is crucial for postnatal skeletal muscle growth, has been investigated widely. Nutrients in ovo were suggested to be critical in embryo muscle development since the chick growth mostly relies on nutrients in eggs at the early developmental stage. Phytosterol esters (PE), which are derived from the reactions between phytosterols and fatty acids, were demonstrated to have important effects on lipid and cholesterol metabolism regulation. In order to reveal the effect of maternal lipid metabolism on the deposition of nutrients in eggs and the development of embryonic muscles, broiler hens were fed with a diet supplemented with 5% PE or control diet. Lipid deposition in eggs and growth of the hatched chicks were studied. We found that PE increased bile acid (BA) deposition in the eggs and serum of hens (p=0.02 and p<0.01, respectively), altered insulin and glucose level differentially in female and male offspring, and promoted body weight (p=0.02 for male and female on day 49), muscle fiber density (p=0.02 for female on day 49), and myogenin and myogenic determination factor (myoD) expression (p=0.03 and p=0.02 on day 49) by the activation of BA receptors in female, but not in male, offspring. Our study determined for the first time that PE promoted muscle development of chicks hatching from eggs laid by the hens, through regulating bile acid (BA) deposition and this may be attributed to the activation of BA receptors.

Ca2+-Calcineurin-NFAT pathway mediates the effect of thymol on oxidative metabolism and fiber-type switch in skeletal muscle.

Thymol is a major component of thyme, and it has been reported that thymol administration reduces body weight, plasma insulin and blood glucose in type-2 diabetes. Skeletal muscle is the most important metabolism organs in the body; however, to date, there is no report on the effect of thymol on skeletal muscle. Our goal was to determine whether thymol has an effect on the different types of skeletal muscle fibers and their metabolism characteristics. Hence, we performed in vivo and in vitro experiments. In vivo, SD rats (4 weeks old) were fed with different concentrations of thymol for 4 weeks, and in vitro C2C12 myotubes were directly treated with thymol for 2 days. The rats fed with 0.025% thymol showed a significantly lower body weight, subcutaneous white adipose tissue index and gastrocnemius muscle index (P < 0.05), while their proportion of brown adipose tissue significantly increased (P < 0.05). The protein and mRNA expression of MyHC I and MyHC IIa in the gastrocnemius muscle of the rats significantly increased (P < 0.05), while the protein level of MyHC II and mRNA expression of MyHC IIb decreased (P < 0.05). Furthermore, 0.025% thymol supplement significantly reduced (P < 0.05) the activity of lactate dehydrogenase (LDH) in the gastrocnemius muscle of the rats, but their succinate dehydrogenase (SDH) and hexokinase (HK) activities increased (P < 0.05). Also, the expression of the fatty acid oxidation-related genes in the gastrocnemius muscle of the rats decreased with the thymol supplement (P < 0.05). In vitro, similar results were obtained. Furthermore, the Ca2+-calcineurin-NFAT pathway, which is an important pathway to regulate the transformation of skeletal muscle fiber type, was studied. We found that the effects of thymol on the myosin heavy chain isoforms, genes related to metabolism and the activation of the Ca2+-calcineurin-NFAT pathway were all reversed by a Ca2+ chelator (P < 0.05). Thus, thymol can promote the oxidative metabolism and fiber type switch in skeletal muscle, and the Ca2+-calcineurin-NFAT pathway plays an important role in it.

Lauric Acid Accelerates Glycolytic Muscle Fiber Formation through TLR4 Signaling.

Lauric acid (LA), which is the primary fatty acid in coconut oil, was reported to have many metabolic benefits. TLR4 is a common receptor of lipopolysaccharides and involved mainly in inflammation responses. Here, we focused on the effects of LA on skeletal muscle fiber types and metabolism. We found that 200 µM LA treatment in C2C12 or dietary supplementation of 1% LA increased MHCIIb protein expression and the proportion of type IIb muscle fibers from 0.452 ± 0.0165 to 0.572 ± 0.0153, increasing the mRNA expression of genes involved in glycolysis, such as HK2 and LDH2 (from 1.00 ± 0.110 to 1.35 ± 0.0843 and from 1.00 ± 0.123 to 1.71 ± 0.302 in vivo, respectively), decreasing the catalytic activity of lactate dehydrogenase (LDH), and transforming lactic acid to pyruvic acid. Furthermore, LA activated TLR4 signaling, and TLR4 knockdown reversed the effect of LA on muscle fiber type and glycolysis. Thus, we inferred that LA promoted glycolytic fiber formation through TLR4 signaling.

Phytol Promotes the Formation of Slow-Twitch Muscle Fibers through PGC-1α/miRNA but Not Mitochondria Oxidation.

Phytol is a side chain of chlorophyll belonging to the side-chain double terpenoid. When animals consume food rich in chlorophyll, phytol can be broken down to phytanic acid after digestion. It was reported that feeding animals with different varieties and levels of forage could significant improve pH and marbling score of steer and lamb carcasses, but the internal mechanism for this is still not reported. The marbling score and pH of muscle was mainly determined by skeletal muscle fiber type, which is due to expression of different myosin heavy-chain (MHC) isoforms. Here, we provide evidence that phytol can indeed affect the diversity of muscle fiber types both in vitro and in vivo and demonstrate that phytol can increase the expression of MHC I (p < 0.05), likely by upgrading the expression of PPARδ, PGC-1α, and related miRNAs. This fiber-type transformation process may not be caused by activated mitochondrial metabolism but by the structural changes in muscle fiber types.

Epigallocatechin Gallate Reduces Slow-Twitch Muscle Fiber Formation and Mitochondrial Biosynthesis in C2C12 Cells by Repressing AMPK Activity and PGC-1α Expression.

Epigallocatechin gallate (EGCG) is a major active compound in green tea polyphenols. EGCG acts as an antioxidant to prevent the cell damage caused by free radicals and their derivatives. In skeletal muscle, exercise causes the accumulation of intracellular reactive oxygen species (ROS) and promotes the formation of slow-type muscle fiber. To determine whether EGCG, as a ROS scavenger, has any effect on skeletal muscle fiber type, we applied different concentrations (0, 5, 25, and 50 µM) of EGCG in the culture medium of differentiated C2C12 cells for 2 days. The fiber-type composition, mitochondrial biogenesis-related gene expression, antioxidant and glucose metabolism enzyme activity, and ROS levels in C2C12 cells were then detected. According to our results, 5 µM EGCG significantly decreased the cellular activity of SDH, 25 µM EGCG significantly downregulated the MyHC I, PGC-1α, NRF-1, and p-AMPK levels and SDH activity while enhancing the CAT and GSH-Px activity and decreasing the intracellular ROS levels, and 50 µM EGCG significantly downregulated MyHC I, PGC-1α, and NRF-1 expression and HK and SDH activity while increasing LDH activity. Furthermore, 300 µM H2O2 and 0.5 mM AMPK agonist (AICAR) improved the expression of MyHC I, PGC-1α, and p-AMPK, which were all reversed by 25 µM EGCG. In conclusion, the effect of EGCG on C2C12 cells may occur through the reduction of the ROS level, thereby decreasing both AMPK activity and PGC-1α expression and eventually reducing slow-twitch muscle fiber formation and mitochondrial biosynthesis.

A missing geographic link in the distribution of the genus Echinotriton (Caudata: Salamandridae) with description of a new species from southern China.

Disjunct geographic distribution of a species or a group of species is the product of long-term interaction between organisms and the environment. Filling the distributional gap by discovery of a new population or a species has significant biogeographic implications, because it suggests a much wider past distribution and provides evidence for the route of range expansion/contraction. The salamandrid genus Echinotriton (commonly known as spiny salamanders, spiny newts, or crocodile newts) has two species that are restricted to two widely separated areas, one in eastern Zhejiang province, China and the other in the Ryukyu Archipelago of Japan. It has been hypothesized that Echinotriton was once continuously distributed between the two areas through a historical land bridge that connected mainland China, Taiwan, and the archipelago. Finding fossils or relic populations along the postulated distribution are strong evidence for the hypothesis. Hundred-twenty-two years after the description of E. andersoni and eight-one years after that of E. chinhaiensis, we discover a third species of Echinotriton in southern China, which fills the distributional gap of the former two species. Species status of the new species is confirmed through molecular phylogenetic analysis and morphological comparison. Mitochondrial DNA indicates that the new species is sister to E. chinhaiensis, while nuclear DNA does not support this relationship. The new species has a very large quadrate projection, a single line of lateral warts pierced by distal rib extremities, normally developed 5th toes, and conical skin tubercles. Our discovery supports the hypothesis that there was a continuous distribution of Echinotriton from eastern coastal China to the Ryukyu Archipelago. We suggest that other species of this genus may also be found in Taiwan. Due to the rarity of this new species, we urge all hobbyists to refrain themselves from collecting this salamander or leaking locality information if encountered, and boycott any trading.

[Diagnosis and treatments for recurrent malignant tumors involving the carotid artery].

OBJECTIVE: To study the diagnosis and treatment of the malignant tumor involving carotid artery. METHODS: A total of 23 cases of recurrent malignant tumors involving the carotid artery were included in this study. For the primary cancers, 8 of 23 cases were laryngeal carcinomas, 10 hypopharyngeal carcinomas, 2 thyroid carcinomas, 1 tonsil carcinoma, 1 parotid gland carcinoma, and 1 hypopharyngeal sarcoma with the invasion of cervical esophagus. Detailed evaluation on each case was performed before treatment. The relations of recurrent tumors with neck blood vessels were determined with enhanced CT/CTA. Of 23 cases with recurrent malignant tumors involving the carotid artery, 16 cases received surgery and 7 cases received the palliative treatment without operation. RESULTS: Seven patients with palliative treatments died of hemorrhage from the invaded neck blood vessels, systemic failure or pulmonary metastasis in six months. Of 16 cases with surgery, recurrent tumors were completely excised in 14 cases and there were residual tumor tissues on artery walls in 2 cases. Within 16 surgical cases, 2 cases died of neck hemorrhoea after one week because of infection, 2 cases died of lung metastasis 8 months later, 3 cases died of neck local recurrence 1 year later, 2 cases died of lung metastasis after 2 years, 1 case died of neck local recurrence 2 years later and 1 case died of a heart attack 2 years later. The rest 5 cases were alive. CONCLUSIONS: Enhancement CT/CTA can used in the evaluation for recurrent malignant tumors involving the carotid artery. Surgical treatments can be applied to some selected patients, which can improve the quality of life and survival time of the patients.

[Swallowing and laryngeal function preserving in surgical treatment of the head and neck tumors involving the tongue root].

OBJECTIVE: To investigate the surgical technique which could preserve the swallowing and laryngeal function effectively in the malignant head and neck tumors involving the tongue root. METHODS: From January 2003 to December 2008, 31 cases of malignant head and neck tumors involving the tongue base had been treated in this hospital were retrospectively analyzed. There were 27 males and 4 females in which 9 cases of primary malignant tumor were from the base of tongue; 3 cases were from the tonsil, 11 cases were from supraglottic laryngeal carcinoma and 8 cases were from hypopharyngeal carcinoma. Preserved the lingual artery of the reserved side and the normal tissue of the root of tongue according to the clinical anatomy of lingual artery during the operation. If preoperative CT had indicated that bilateral lingual arteries were involved, total glossectomy should have been done. The epiglottis, vocal cords and the ventricular band of larynx was preserved as much as possible for the mechanisms of laryngeal function. RESULTS: In this group, residual tongue necrosis did not occurred. One case with total glossectomy didn't remove the trachea cannula. Five had total laryngectomy. The other 25 cases decannulated from 14th days to 90th days postoperatively. The time of oral feeding was started from 10th days to 31st days postoperatively. Two cases with hypopharyngeal carcinoma developed fistula, which were cured by dressing change. Two with root of tongue cancer and 1 with tonsil cancer had postoperative infection and healed in 2 weeks. The median follow-up time was 36 months, and the Kaplan-Meier 3-years and 5-years survival rates were 79.5% and 69.6% respectively. CONCLUSIONS: In the surgical treatments of the malignant head and neck tumors involving the base of tongue, the excisions and reconstructions of the primary tumor and the involved tongue base according to the clinical anatomy of lingual artery and the protection mechanisms of laryngeal function during the operation was one of the most effective technique to preserve the swallowing and laryngeal function.

[Application of lateral cervical incision in the removal of the cervical esophageal foreign body].

OBJECTIVE: To study the indication and clinical application of lateral neck incision for the removal of cervical esophageal foreign bodies. METHODS: From January 1999 to January 2009, 2189 cases esophageal foreign bodies were treated. The clinical data of 137 cases (6.25%) with lateral neck incision were retrospectively analyzed. In these 137 cases, 114 cases were over 16-years-old (adult group), while 23 cases were under 16-years-old (children group). In adult group, 67 cases (58.8%) had esophageal perforation (esophageal perforation with neck abscess 29 cases, esophageal perforation without neck abscess 38 cases); 47 cases did not have esophageal perforation (impacted foreign body without neck abscess 40 cases, foreign body with esophageal abscess 7 cases). In children group, 19 cases (82.6%) had esophageal perforation including 15 cases with neck abscess; 4 cases without esophageal perforation, 3 cases had esophageal abscess and one case without abscess but of huge foreign body. RESULTS: All 137 patients with foreign body were cured through lateral neck incision. Nineteen cases (13.9%) had hoarseness and recovered in 3 months. Five adult patients had post-operative cicatricial stricture of the esophagus, but it was mild and completely recovered by the treatment of dilatation in 3 to 11 months. Nine adult patients with esophageal perforation were cured by secondary suture, the remaining esophageal perforation cases were healed by first intention. One case with common carotid artery impairement by the foreign body was successfully treated by carotid artery ligation without hemiplegia, aphasia and other sequelae. Two cases had cardiopulmonary arrest, 2 cases had febrile convulsions and 1 case had acute respiratory failure, 5 cases had septic shock, all these patients were effectively controlled and cured. Seven of the 9 cases with tracheotomy had the tracheal tube removed during hospitalization; 1 of the two obese patients had the extubation 3 months after the discharge and the other one still had the tube. All esophageal perforation cases in children group had primary healing by perforation apposition suture. Four of the 5 children had successful tracheotomy decannulation, one child had extubation by 2 months through continuously reduced tracheal tube model. CONCLUSION: Penetrating esophageal foreign body and neck abscess were indication of the lateral neck incision, and positive prevention and cure the complications of lateral neck incision could achieve good curative effect.

[A clinical study on improving phonation function of larynx after partial laryngectomy].

OBJECTIVE: To analyse the clinical results of reconstruction of vocal cord using sternohyoid muscle flap after partial laryngectomy and research a new repairing method to improve laryngeal function and living quality of patient. METHOD: The pronunciation of larynx is on the basic of the vibration of the vocal cord by air current. The laryngeal appearance and the function of opposite side vocal cord is very important for improving the quality of pronunciation. We developed a technique of thyroid cartilage membrane and sternohyoid muscle flap to repair the defect left by a vertical laryngectomy and reconstructed the vocal cord. The thyroid cartilage membrane was used to bridge over the upper part defect of laryngeal cavity, the sternohyoid muscle flap forming the opposite side was made a 90 degree bent toward the laryngeal cavity to repaired the lower part defect of laryngeal cavity and reconstruct a new vocal cord. The repaired larynx recovered the normal appearance. The new vocal cord participated speech. The anterior 2/3 part of both vocal cored could contacted in pronunciation of patient. The phonation function of larynx was restored. RESULT: Sixty-one cases partial laryngectomy was taken including vertical partial laryngectomy in 37 cases, extended vertical partial laryngectomy in 24. During their operations, the laryngeal defect was repaired with thyroid cartilage membrane and sternohyoid muscle flap, and a vocal cord was reconstructed in the same time. After operation, the laryngeal cavity repaired has a normal appearance, and the laryngeal satisfactory phonation effect has been obtained. 93.4% of the patients enjoyed a socially acceptable voice and rejoined normal working. CONCLUSION: A technique of thyroid cartilage and sternohyoid muscle flap to repair the defect and reconstructive vocal cord after partial laryngectomy make patient recover normal laryngeal appearance and obtain satisfactory phonation effect. Among the method of repairation for partial laryngectomy sternohyoid muscle flap should be first considered owing to its various merits: 1. abundant material. 2. convenient procedures. 3. smaller trauma. 4. satisfactory effect.

[The surgical treatment of cancer in the base of tongue].

OBJECTIVE: To seek for a better approach for the resection of carcinoma of the base of tongue. METHOD: From 1991 to 2000, all 21 cases of tongue base carcinoma had been removed by transhyoid pharyngotomy(11 cases), mandibulectomy(7 cases) and step-liked mandibulotomy (3 cases). RESULT: Three patients with step-liked mandibulotomy had speech disorder and dysphagy. The rest had satisfactory speech and swallowing functions. During followed-up of 2-5 years, 2 cases lost follow-up, 18 cases were alive at 2 years, 13 cases were alive at 3 years, 5 cases 5 years. CONCLUSION: Transhyoid pharyngotomy provides good exposure to resect those tumors that involve the base of tongue, epiglottis, hypopharynx and larynx.

[Anatomical landmarks of skull base and safety of the skull base surgery].

OBJECTIVE: In order to improve the safety of skull base surgery and to completely resect the skull base tumors, the anatomical landmarks of skull base were studied. METHODS: 29 cases of skull base surgery were performed between 1992 and 2002, and their clinical data were retrospectively analysed. The anatomical landmarks of cranial base, such as comb, pterygoid process, spine of temporal bone, styloid process, and internal caroid artery, were analysed in preoperative diagnosis and operative treatment of various kinds of skull base tumors. RESULTS: In the early stages, because of lacking the knowledge of anatomical landmarks of the skull base, the surgery lasted longer, part of skull base tumors remained and operative blood lose was much more than that in later stage. In the later stage, no operative death and severe complications were found in 26 cases which underwent various kinds of skull base surgery. The survival rates at 3 and 5 years were 72.2% and 35.7% respectively for malignant tumors. CONCLUSIONS: Comb, pterygoid process, spine of temporal bone, styloid process, internal caroid artery and their adjacent structures were important anatomical landmarks for operative treatment of anterior, middle and lateral cranial base tumors. It was important to know the anatomical landmarks to ensure the safety of the skull base surgery.