Objective To explore the effect and mechanism of massage on skeletal muscle overuse injury in rats.Methods Thirty-two male Sprague-Dawley rats were randomly assigned to the blank(8 rats)and modeling groups(24 rats)using a random number table method.A model of skeletal muscle overuse injury was induced in the modeling group using downhill running centrifugal contraction exercise for 4 weeks.The successfully modeled rats were divided into a model group,a massage group,and a Yunnan Baiyao Aerosol group,with 8 rats in each group.The 4 groups of rats were subjucted to balance beam test.After 24 h of completing the modeling,the massage group was treated with a self-made massager,while the Yunnan Baiyao Aerosol group was treated with Yunnan Baiyao Aerosol twice a day for 3 consecutive days.Twenty-four hours after the end of treatment,the four groups of rats underwent behavioral evaluation again using the beam balance test.Hematoxylin-eosin staining was used to observe damage to the gastrocnemius muscle.The enzyme-linked immunosorbent assay was used to measure the concentrations of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and 5-hydroxytryptamine(5-HT)in plasma.Western blotting was used to detect the protein expression levels of phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK),phosphorylated c-Jun N-terminal kinase(p-JNK),and phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2)proteins in the MAPK signaling pathway.Results Before treatment,compared to the blank group,the rats in the model group,massage group,and Yunnan Baiyao Aerosol group had a longer walking time on the balance beam and a higher number of sliding claws(P<0.05).After treatment,compared with the blank group,the model group showed longer walking time on the balance beam,increased number of sliding claws,significant infiltration of inflammatory cells in gastrocnemius muscle,increased levels of TNF-α,IL-6,5-HT,and p-p38 MAPK,p-JNK,and p-ERK1/2(P<0.05).Compared to the model group,the walking time of the balance beam in the massage group was shortened,the number of sliding claws was reduced,the number of inflammatory cells was reduced,TNF-α,IL-6,and 5-HT indices decreased,and p-p38 MAPK,p-JNK,p-ERK1/2 indices decreased(P<0.05).The Yunnan Baiyao Aerosol group showed a decrease in the number of sliding claws,fewer inflammatory cells,reduced IL-6 levels,and decreased p-JNK and p-ERK1/2(P<0.05).Compared to the massage group,the Yunnan Baiyao Aerosol group had more inflammatory cells,increased IL-6 levels,and elevated p-JNK and p-ERK1/2 levels(P<0.05).Conclusion Massage can improve chronic skeletal muscle fatigue injury,and its mechanism of action may be through inhibiting the MAPK signaling pathway,downregulating the p-ERK1/2,p-p38 MAPK,and p-JNK proteins in this pathway,indirectly affecting the release of inflammatory factors,thus reducing skeletal muscle cell damage,inhibiting the release of inflammatory mediators,and promoting skeletal muscle repair.

Objective To explore the effect and mechanism of massage on skeletal muscle overuse injury in rats.Methods Thirty-two male Sprague-Dawley rats were randomly assigned to the blank(8 rats)and modeling groups(24 rats)using a random number table method.A model of skeletal muscle overuse injury was induced in the modeling group using downhill running centrifugal contraction exercise for 4 weeks.The successfully modeled rats were divided into a model group,a massage group,and a Yunnan Baiyao Aerosol group,with 8 rats in each group.The 4 groups of rats were subjucted to balance beam test.After 24 h of completing the modeling,the massage group was treated with a self-made massager,while the Yunnan Baiyao Aerosol group was treated with Yunnan Baiyao Aerosol twice a day for 3 consecutive days.Twenty-four hours after the end of treatment,the four groups of rats underwent behavioral evaluation again using the beam balance test.Hematoxylin-eosin staining was used to observe damage to the gastrocnemius muscle.The enzyme-linked immunosorbent assay was used to measure the concentrations of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and 5-hydroxytryptamine(5-HT)in plasma.Western blotting was used to detect the protein expression levels of phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK),phosphorylated c-Jun N-terminal kinase(p-JNK),and phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2)proteins in the MAPK signaling pathway.Results Before treatment,compared to the blank group,the rats in the model group,massage group,and Yunnan Baiyao Aerosol group had a longer walking time on the balance beam and a higher number of sliding claws(P<0.05).After treatment,compared with the blank group,the model group showed longer walking time on the balance beam,increased number of sliding claws,significant infiltration of inflammatory cells in gastrocnemius muscle,increased levels of TNF-α,IL-6,5-HT,and p-p38 MAPK,p-JNK,and p-ERK1/2(P<0.05).Compared to the model group,the walking time of the balance beam in the massage group was shortened,the number of sliding claws was reduced,the number of inflammatory cells was reduced,TNF-α,IL-6,and 5-HT indices decreased,and p-p38 MAPK,p-JNK,p-ERK1/2 indices decreased(P<0.05).The Yunnan Baiyao Aerosol group showed a decrease in the number of sliding claws,fewer inflammatory cells,reduced IL-6 levels,and decreased p-JNK and p-ERK1/2(P<0.05).Compared to the massage group,the Yunnan Baiyao Aerosol group had more inflammatory cells,increased IL-6 levels,and elevated p-JNK and p-ERK1/2 levels(P<0.05).Conclusion Massage can improve chronic skeletal muscle fatigue injury,and its mechanism of action may be through inhibiting the MAPK signaling pathway,downregulating the p-ERK1/2,p-p38 MAPK,and p-JNK proteins in this pathway,indirectly affecting the release of inflammatory factors,thus reducing skeletal muscle cell damage,inhibiting the release of inflammatory mediators,and promoting skeletal muscle repair.

ObjectiveTo explore the activating effects of ten important effective components in seven medicinal and edible substances on human pregnane X receptor （PXR）， including Glycyrrhizae Radix et Rhizoma （liquiritin and glycyrrhizic acid）， Houttuyniae Herba （quercetin and houttuyfonate）， Prunellae Spica （rosmarinic acid）， Cassiae Semen （aurantio-obtusin）， Poria （pachymic acid）， Lilii Bulbus （Lilium brownii saponin and colchicine）， and Lycii Fructus （Lycium barbarum polysaccharide） and screen potentially toxic components. MethodCell counting kit-8 （CCK-8） assay was used to investigate the cytotoxic effect of liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， pachymic acid， aurantio-obtusin， and colchicine （10， 20， and 50 μmol·L^-1）， and L. brownii saponin and L. barbarum polysaccharide （10， 20， and 50 mg·L^-1） on normal human hepatocyte cell line （L02）. The release of lactate dehydrogenase （LDH） in L02 cells after drug treatments was detected by the biochemical analyzer. The apoptosis induced by ten effective components was explored by Hoechst 33342 staining. The secreted luciferase reporter system was used to co-transfect the PXR expression vector and reporter gene vector containing cytochrome P450 3A4 （CYP3A4） transcriptional regulatory region into L02 cells， with 10 μmol·L^-1 rifampicin （RIF） as a positive control. After treated with liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， aurantio-obtusin， pachymic acid， and colchicine （5， 10， and 20 μmol·L^-1） and L. brownii saponin and L. barbarum polysaccharide （5， 10， and 20 mg·L^-1） for 24 h， the cells were tested for secreted luciferase activity. ResultCompared with the control group， colchicine， L. brownii saponin， and quercetin decreased the cell viability （P<0.05， P<0.01）. Compared with the control group， quercetin， rosmarinic acid， glycyrrhizic acid， colchicine， aurantio-obtusin， and pachymic acid increased the release rate of LDH in L02 cells （P<0.05， P<0.01）. The proportion of hyperchromatic nuclei increased gradually after rosmarinic acid， liquiritin， and L. barbarum polysaccharide treatments as compared with the control group （P<0.05， P<0.01）. In terms of co-transfection of pcDNA3.1-PXR and pGLuc-CYP3A4 into L02 cells， compared with the control group， aurantio-obtusin and pachymic acid showed activating effects on PXR （P<0.05）， whereas liquiritin and glycyrrhizic acid showed inhibitory effects （P<0.05）. ConclusionThe findings suggest that when medicinal and edible substances are taken for a long time， attention should be paid to their influence on drug-metabolizing enzymes and possible interactions， so as to improve their safety.

Objective: To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan. Methods: We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected. Results: We have expressed and purified vaccinia virus proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early protein E3 and late protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of late protein F17 was blocked detected by Western blot. Conclusions The expression limitation of late protein F17 may be an explanation for the replication-defective mechanism of NTV.

Objective: To investigate the cellular and humoral immune responses induced by combined immunization with the fusion protein of human papillomavirus type 18 (HPV18) and the recombinant vaccinia virus. Methods: Purified HPV18L2_31-600E7E6 fusion protein, expressed by prokaryotic expression system, were immunized in combination with the recombinant vaccinia virus vaccine expressing HPV18E7E6 fusion protein (rVV18E7E6) by using various prime-boost regiments in C57BL/6 mice. Cellular and humoral immune responses were analyzed by enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and pseudovirus neutralization assay. Results: Higher levels of cellular immune responses were induced in mice primed with the HPV18L2_31-600E7E6 fusion protein/adjuvant CpG and boosted with the recombinant vaccinia virus rVV18E7E6, than in other immunized mice. Higher binding antibody level was induced, and low level neutralizing antibody against pseudovirus was detected simultaneously. Conclusions Priming with HPV18L2_31-600E7E6 fusion protein/CpG and boosting with the recombinant vaccinia virus rVV18E7E6 could induce higher cellular and humoral immune response in immunized mice, which might be taken as vaccine candidate for treatment of HPV18 chronic infection and postoperative adjuvant treatment for cervical cancer.

Objective: To generate monkeypox virus specific monoclonal antibodies for further establishing monkeypox virus immunofluorescence assay. Methods: Monkeypox virus A29 protein, vaccinia ortholog A27 protein and cowpox ortholog 162 protein were expressed in E. coli BL21 to screen antibodies. Synthetic monkeypox virus A29_{17 ~ 49} polypeptide was used to immune BALB/c mice. Monkeypox virus monoclonal antibodies were generated through fusion, cloning and screening techniques. Indirect ELISA was performed to test antibodies specificity and subtype. Results: A29, A27 and 162 proteins were highly expressed in E. coli and detected by Western blot. The three his-tagged proteins were purified using His-Bind affinity chromatography column. The purity of the proteins was all more than 90%. And 8 strains monkeypox virus specific monoclonal antibodies were screened by the three purified proteins. Two mAbs of 8 were IgG3 subtype and the rest were IgG1 subtype. Conclusions Eight strains of monkeypox virus specific monoclonal antibodies were generated, they can be used to further establish monkeypox virus immune immunofluorescence assay.