ABSTRACT
As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (Km = 0.263 mM, kcat/Km = 562.3 s-1 mM-1 for p-nitrophenyl acetate (pNPA2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstFP144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstFP144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstFP144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes.
ABSTRACT
Acupuncture pretreatment (AP) has a good skeletal muscle protective effect. The present study investigated whether acupuncture pretreatment could improve ultrastructural changes and skeletal muscle inflammation in exercising skeletal muscle injury. Eighty-eight male SD rats were randomly divided into a blank group (C), an exercise group (E), and an acupuncture pretreatment group (AP). Among them, the E and AP groups were divided into five subgroups of 0h, 12h, 24h, 48h, and 72h according to the extraction time after exercise, with 11 groups and eight rats in each group. The study involved simulating skeletal muscle injury caused by intermittent downhill running centrifugal exercise. The researchers used various methods to observe changes in mitochondrial structure and cGAS-STING-NF-κB p65 protein content of classical inflammatory response signaling pathway. These methods included transmission electron microscopy to observe skeletal muscle, Western Blot to detect changes in protein content, and reverse transcription polymerase chain reaction (RT-qPCR method) to detect cytoplasmic mtDNA gene fragment ND1, D-LOOP and cGAS-STING- NF-κB p65 protein RNA. The aim was to investigate the changes in NF-κB p65 protein RNA. Changes in NF-κB p65 protein RNA content and mtDNA gene fragment ND1 and D-LOOP content; changes in serum IL-8 and IFN-ß content were detected by enzyme-linked immunosorbent assay (ELISA); WB, RT-qPCR, ELISA assays aimed to study the skeletal muscle injury and mitochondrial structural damage in group E relationship skeletal muscle tissue level, cytoplasmic mtDNA fragment gene ND1, and D-LOOP content in skeletal muscle tissue of group E. In comparison to group C, the levels of cGAS-STING-NF-κB p65 protein expression and mRNA, and the serum levels of IL-8 and IFN-ß were significantly higher in group E . However, the acupuncture pretreatment group (AP) reduced the extent of damage to skeletal muscle mitochondria and the levels of cytoplasmic mtDNA fragment genes ND1 and D-LOOP. Also, the high expression of cGAS-STING-NF-κB p65 protein, mRNA, and the levels of IL-8 and IFN-ß were inhibited in the AP group. The results indicated that AP ameliorated exercise-induced skeletal muscle injury and reduced skeletal muscle inflammation produced after centrifugal exercise. This was achieved by inhibiting the overexpression of the cGAS-STING-NF-κB signaling pathway.
ABSTRACT
Bibliometric and scientific knowledge graph methods were used to analyze the research status and hot spots of acupuncture-moxibustion in treatment of myofascial pain syndrome (MPS) and explore its development trend. The articles of both Chinese and English versions relevant to MPS treated by acupuncture-moxibustion were searched in CNKI, VIP, Wanfang, SinoMed and WOS from the database inception to March 20, 2023. Using Excel2016, CiteSpace6.2.R2 and VOSviewer1.6.18, the visual analysis was conducted by means of the cooperative network, keyword co-occurrence, keyword timeline, keyword emergence, etc. From Chinese databases and WOS database, 910 Chinese articles and 300 English articles were included, respectively. The annual publication volume showed an overall rising trend. Literature output of English articles was concentrated in Spain, China, and the United States, of which, there was less cross-regional cooperation. In the keyword analysis, regarding acupuncture-moxibustion therapy, Chinese articles focused on "acupuncture", "electroacupuncture" and "acupotomy"; while, "dry needling" and "injection" were dominated for English one. Clinical study was the current hot spot in Chinese databases, in comparison, the randomized controlled double-blind clinical trial was predominant in WOS. Both Chinese and English articles were limited in the report of mechanism research. The cooperation among research teams should be strengthened to conduct comparative research, dose-effect research and effect mechanism research with different methods of acupuncture-moxibustion involved so that the evidences can be provided for deeper exploration.
Subject(s)
Acupuncture Therapy , Electroacupuncture , Moxibustion , Myofascial Pain Syndromes , Humans , Pattern Recognition, Automated , Myofascial Pain Syndromes/therapyABSTRACT
OBJECTIVES: To observe the effects of acupuncture pretreatment on toll-like receptor 9/myeloid differentiation factor 88/nuclear factor-κB (TLR9/MyD88/NF-κB) signaling pathway and inflammatory response in the rats with exercise-induced skeletal muscle damage (EIMD) and explore the underlying mechanism of this pretreatment for EIMD. METHODS: A total of 88 male SD rats were randomly divided into a blank group (8 rats), a model group (40 rats) and an acupuncture pretreatment group (40 rats). In the model group and the acupuncture pretreatment group, 5 subgroups were randomized according to the sampling time of 0, 12, 24, 48 and 72 h of modeling, with 8 rats in each one, respectively. Before modeling, in the acupuncture pretreatment group, acupuncture was delivered at bilateral "Zusanli" (ST 36) and "Guanyuan" (CV 4) for 20 min, once daily for consecutive 7 days. By one-time intermittent downhill centrifugal exercise on animal experimental treadmill, EIMD model was established in the model group and the acupuncture pretreatment group. The ultrastructure of gastrocnemius muscle was observed under transmission electron microscope. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum were detected by ELISA. The protein and mRNA expression of TLR9, MyD88 and NF-κB p65 in the gastrocnemius tissue of rats was detected by the Western blot and real-time PCR, respectively. RESULTS: Compared with the blank group, the gastrocnemius ultrastructure in the model group showed the damage of different degrees, with myofilaments disarranged and twisted, mitochondria obviously swollen and mitochondrial crista partially defected. Compared with the model group, the injury was mild, most of muscle fibers were arranged neatly and the number of mitochondria increased remarkably in the acupuncture pretreatment group. Compared with the blank group, in the model group, the serum IL-6 levels increased at 0, 12, 24 and 48 h after modeling in the rats (P<0.01), and TNF-α levels were elevated at each time point after modeling (P<0.05, P<0.01). In the acupuncture pretreatment group, the serum IL-6 levels were reduced at 12, 24 and 48 h after modeling (P<0.01, P<0.05), and the TNF-α levels decreased at 24, 48 and 72 h after modeling (P<0.01) when compared with those in the model group at the same time points separately. The serum IL-6 and TNF-α levels ascended and then tended to decline in the model group and the acupuncture pretreatment group. Compared with the blank group, the protein and mRNA expression of TLR9 and NF-κB p65 in the gastrocnemius tissue increased at 12, 24, 48 and 72 h after modeling (P<0.01, P<0.05), and the protein and mRNA expression levels of MyD88 in the gastrocnemius tissue at each time point after modeling were elevated (P<0.05, P<0.01) in the model group. When compared with the model group at the same time points, in the acupuncture pretreatment group, the protein expression of TLR9 in the gastrocnemius tissue decreased at 12, 24, 48 and 72 h after modeling (P<0.05, P<0.01), and the mRNA expression of TLR9 was declined at 24, 48 and 72 h after modeling (P<0.01, P<0.05); the protein and mRNA expression of MyD88 in the gastrocnemius decreased at 24, 48 and 72 h after modeling (P<0.01, P<0.05), and that of NF-κB p65 was reduced at 24 h and 48 h after modeling (P<0.01). The protein and mRNA expression of TLR9, MyD88 and NF-κB p65 in the gastrocnemius tissue showed a trend of decrease after increase in the model group and the acupuncture pretreatment group. CONCLUSIONS: Acupuncture pretreatment can alleviate exercise-induced skeletal muscle damage, which may be related to modulating the expression of TLR9/MyD88/NF-κB signaling pathway to inhibit the inflammatory response.
