ObjectiveTo make clear exercise combined with Shenghui Tang interferes in acetylcholine receptor （M1AChR） to improve mitochondrial autophagy and enhance cognition of Alzheimer's disease （AD） model rats through the adenylate activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsForty-eight male SD rats of SPF grade were randomly divided into a blank group， a model group， a Shenghui Tang group （9.3 g·kg^-1）， an exercise group， an exercise + Shenghui Tang group （9.3 g·kg^-1）， and a rapamycin group （1.5 mg·kg^-1）. Except for the blank group， the rat model of AD was constructed by injecting amyloid beta （Aβ_1-42） into hippocampus stereotaxically. The exercise group received treadmill exercise for 4 weeks， while the Shenghui Tang group received intragastric administration for 4 weeks， and the exercise + Shenghui Tang group received treadmill exercise and intragastric administration of Shenghui Tang for 4 weeks simultaneously. After the intervention， the Morris water maze test was used to detect the learning and memory ability. Spontaneous behavior was observed in the open field test. The pathological structure of hippocampal neurons was observed by NISSl staining. The expression level of M1AChR in hippocampus was detected by immunohistochemistry （IHC）. The autophagy ultrastructure of hippocampal neurons was observed by transmission electron microscopy. The apoptosis rate was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）. The expression of Beclin1 and microtubule-associated protein light chain 3β （LC3β） was detected by immunofluorescence （IF）. The protein expression of M1AChR， AMPK， p-AMPK， mTOR， Beclin1， LC3β， and chelate 1 （SQSTM1/p62） in hippocampus was detected by Western blot. ResultsCompared with the blank group， the model group exhibited significantly increased platform escape latency on the fifth day （P<0.01） and significantly decreased activity distance in the target quadrant and times of crossing the platform （P<0.01）. The total movement distance in the open field， the time of movement in the central area， and the average speed obviously decreased （P<0.05）. The arrangement of nerve cells in hippocampus CA1 region was dispersed， and the numbers of Nissl bodies and M1AChR positive cells significantly decreased （P<0.01）. The expression of TUNEL positive cells was significantly increased （P<0.01）. The typical autophagic lysosomal structure decreased. The protein expression of M1AChR， p-AMPK/AMPK， p-mTOR/mTOR， Beclin1， LC3Ⅱ/Ⅰ in hippocampus was significantly decreased （P<0.01）， and the protein expression of p62 was significantly increased （P<0.01）. Compared with the model group， the exercise + Shenghui Tang group exhibited obviously improved space exploration and positioning navigation ability （P<0.05， P<0.01）. The total movement distance in the open field， the time of movement in the central area， and the average speed of movement significantly increased （P<0.01）. The number of Nissl bodies significantly increased （P<0.01）. The number of M1AChR positive cells in hippocampus was significantly increased （P<0.01）. The expression of TUNEL positive cells was significantly decreased （P<0.01）. The protein expression of M1AChR， p-AMPK/AMPK， p-mTOR/mTOR， Beclin1， LC3Ⅱ/Ⅰ in hippocampus was significantly increased （P<0.01）， while the protein expression of p62 was significantly decreased （P<0.01）. Compared with the exercise + Shenghui Tang group， the Shenghui Tang group and the exercise group showed significantly increased platform escape latency on the fifth day （P<0.01） and obviously decreased activity distance in the target quadrant and times of crossing the platform （P<0.05， P<0.01）. The total movement distance in the open field， the time of movement in the central area， and the average speed of movement significantly decreased （P<0.01）. The number of Nissl bodies and the number of M1AChR positive cells significantly decreased （P<0.01）. The expression of TUNEL positive cells was obviously increased （P<0.05）. Ultrastructure of the hippocampal region showed decreased autophagy level. The protein expression of M1AChR， p-AMPK/AMPK， p-mTOR/mTOR， LC3Ⅱ/Ⅰ in the hippocampus was obviously decreased in the Shenghui Tang group （P<0.05， P<0.01）， while the protein expression of p62 was significantly increased （P<0.01）. In the exercise group， the protein expression of M1AChR， p-AMPK/AMPK， Beclin1， and LC3Ⅱ/Ⅰ was obviously decreased （P<0.05， P<0.01）， while the protein expression of p-mTOR/mTOR and p62 was significantly increased （P<0.01）. ConclusionExercise combined with traditional Chinese medicine can enhance the expression of M1AChR in the hippocampus of AD model rats， induce autophagy through the AMPK/mTOR signaling pathway， and improve the learning and memory ability of AD rats.

ObjectiveThis paper aims to investigate the effects of icariin on spermatogenesis in mice with exercise-induced fatigue and explore the underlying mechanisms. MethodsICR male mice were screened by swimming and randomly divided into normal group， model group， vitamin C group， icariin groups with low， medium， and high doses， and medium-dose icariin+N-nitro-L-arginine methyl ester （L-NAME） group， with 10 mice per group. Except for the normal group， all the other groups underwent weighted swimming training to establish an exercise-induced fatigue model. No gavage was administered during the first two weeks of the weighted training. From week three to four， the icariin groups with low， medium， and high doses received 0.03， 0.06， and 0.12 g·kg^-1 icariin via gavage， respectively. The vitamin C group received 0.2 g·kg^-1 vitamin C. The L-NAME group received 0.06 g·kg^-1 icariin and 0.01 g·kg^-1 L-NAME via intraperitoneal injection. The normal and model groups received equivalent physiological saline. After the experiment， body weight and the last exhaustive swimming time were recorded. Blood urea nitrogen （BUN）， lactate （LA）， lactate dehydrogenase （LDH）， malondialdehyde （MDA）， testicular testosterone （T）， testicular Ca²⁺/Mg²⁺-adenosine triphosphatase （ATPase） （micro-assay）， and the levels of testicular cyclic guanosine monophosphate （cGMP） were measured by using kits. Sperm CD46 levels were detected by flow cytometry. Testicular seminiferous tubules were observed via hematoxylin-eosin （HE） staining， and the testicular morphometric score （TMS） was used to evaluate the spermatogenic function. Protein expression of regucalcin （RGN， SMP30）， cGMP-dependent protein kinase 1 （PKG）， and cGMP-dependent protein kinase anchoring protein （GKAP1） was detected by Western blot. Testicular regucalcin expression was examined by immunofluorescence （IF）. The epididymal sperm quality of mice was observed under a microscope. Fluorescence-stained sections of stimulated by retinoic acid gene 8 （STRA8）， synaptonemal complex protein 3 （SCP3）， and transition protein 1（TNP1） in testicular seminiferous tubules were assessed by immunohistochemistry （IHC）. ResultsCompared with the normal group， the model group showed decreased body weight and exhaustive swimming time （P<0.01）， significantly increased fatigue markers （LA， LDH， and BUN） and lipid peroxidation product MDA （P<0.01）， reduced testicular RGN， PKG， GKAP1， testosterone， Ca²⁺/Mg²⁺-ATPase， and cGMP levels （P<0.01）， decreased sperm motility， sperm count， and TMS scores， and downregulated the expression of STRA8， SCP3， and TNP1. Compared with the model group， the icariin group with high dose exhibited increased exhaustive swimming time （P<0.01）， reduced LA， LDH， BUN， and MDA levels （P<0.01）， elevated superoxide dismutase （SOD） （P<0.01）， upregulated testicular RGN， PKG， GKAP1， testosterone， Ca²⁺/Mg²⁺-ATPase， and cGMP levels （P<0.01）， improved sperm motility， sperm count， and TMS scores， and enhanced STRA8， SCP3， and TNP1 expression. Compared with the L-NAME group， the icariin group with medium dose showed increased expression of STRA8， SCP3， and TNP1 in the testicular tissue （P<0.01） and elevated cGMP and GKAP1 levels （P<0.01）. ConclusionExercise-induced fatigue reduces the expression of RGN and cGMP/PKG/GKAP1 in mice， thereby causing abnormal spermatogenesis and impairing reproductive function in mice. Icariin ameliorates spermatogenic dysfunction in exercise-induced fatigue mice by promoting the expression of RGN and cGMP/PKG/GKAP1， thereby mitigating the damage of exercise-induced fatigue to the reproductive system.

ObjectiveThis paper aims to investigate the effects of icariin on spermatogenesis in mice with exercise-induced fatigue and explore the underlying mechanisms. MethodsICR male mice were screened by swimming and randomly divided into normal group， model group， vitamin C group， icariin groups with low， medium， and high doses， and medium-dose icariin+N-nitro-L-arginine methyl ester （L-NAME） group， with 10 mice per group. Except for the normal group， all the other groups underwent weighted swimming training to establish an exercise-induced fatigue model. No gavage was administered during the first two weeks of the weighted training. From week three to four， the icariin groups with low， medium， and high doses received 0.03， 0.06， and 0.12 g·kg^-1 icariin via gavage， respectively. The vitamin C group received 0.2 g·kg^-1 vitamin C. The L-NAME group received 0.06 g·kg^-1 icariin and 0.01 g·kg^-1 L-NAME via intraperitoneal injection. The normal and model groups received equivalent physiological saline. After the experiment， body weight and the last exhaustive swimming time were recorded. Blood urea nitrogen （BUN）， lactate （LA）， lactate dehydrogenase （LDH）， malondialdehyde （MDA）， testicular testosterone （T）， testicular Ca²⁺/Mg²⁺-adenosine triphosphatase （ATPase） （micro-assay）， and the levels of testicular cyclic guanosine monophosphate （cGMP） were measured by using kits. Sperm CD46 levels were detected by flow cytometry. Testicular seminiferous tubules were observed via hematoxylin-eosin （HE） staining， and the testicular morphometric score （TMS） was used to evaluate the spermatogenic function. Protein expression of regucalcin （RGN， SMP30）， cGMP-dependent protein kinase 1 （PKG）， and cGMP-dependent protein kinase anchoring protein （GKAP1） was detected by Western blot. Testicular regucalcin expression was examined by immunofluorescence （IF）. The epididymal sperm quality of mice was observed under a microscope. Fluorescence-stained sections of stimulated by retinoic acid gene 8 （STRA8）， synaptonemal complex protein 3 （SCP3）， and transition protein 1（TNP1） in testicular seminiferous tubules were assessed by immunohistochemistry （IHC）. ResultsCompared with the normal group， the model group showed decreased body weight and exhaustive swimming time （P<0.01）， significantly increased fatigue markers （LA， LDH， and BUN） and lipid peroxidation product MDA （P<0.01）， reduced testicular RGN， PKG， GKAP1， testosterone， Ca²⁺/Mg²⁺-ATPase， and cGMP levels （P<0.01）， decreased sperm motility， sperm count， and TMS scores， and downregulated the expression of STRA8， SCP3， and TNP1. Compared with the model group， the icariin group with high dose exhibited increased exhaustive swimming time （P<0.01）， reduced LA， LDH， BUN， and MDA levels （P<0.01）， elevated superoxide dismutase （SOD） （P<0.01）， upregulated testicular RGN， PKG， GKAP1， testosterone， Ca²⁺/Mg²⁺-ATPase， and cGMP levels （P<0.01）， improved sperm motility， sperm count， and TMS scores， and enhanced STRA8， SCP3， and TNP1 expression. Compared with the L-NAME group， the icariin group with medium dose showed increased expression of STRA8， SCP3， and TNP1 in the testicular tissue （P<0.01） and elevated cGMP and GKAP1 levels （P<0.01）. ConclusionExercise-induced fatigue reduces the expression of RGN and cGMP/PKG/GKAP1 in mice， thereby causing abnormal spermatogenesis and impairing reproductive function in mice. Icariin ameliorates spermatogenic dysfunction in exercise-induced fatigue mice by promoting the expression of RGN and cGMP/PKG/GKAP1， thereby mitigating the damage of exercise-induced fatigue to the reproductive system.

Aim To systematically elucidate the molecular regulatory network of thermogenic function in brown adipose tissue(BAT)through multi-omics integrative analysis,to discover novel thermogenic regulatory genes and provide novel therapeutic targets for metabolic disorders.Methods A novel methodology for screening key genes regulating thermogenesis in BAT was constructed:First,differential expression analysis was performed on bulk RNA-seq data from murine BAT.Genes meeting the thresholds of ABS(log2FoldChange)＞1 and Padj＜0.05 were identified as differentially expressed genes.Intersectional analysis was then applied to obtain consensus upregulated and downregulated gene sets.Subsequently,scRNA-seq data of brown adipocytes were partitioned into high-expression group and low-expression group based on the expression levels of candidate genes.Differential analysis and gene set enrichment analysis(GSEA)were conducted between these groups to assess the correlation between candidate genes and thermogenic function.Finally,ex-perimental validation of selected candidate genes was performed using quantitative real-time PCR and Western blot.Results Bioinformatics analysis identified 65 thermogenesis-positive correlated genes and 7 thermogenesis-negative corre-lated genes.Subsequent quantitative PCR validation demonstrated that candidate genes Mfsd2a,Me1,Slc25a34,Pfkp,Ankrd9,Hsd17b12,Aldoa,Ctsz and Pcyt2 exhibited upregulation exceeding 5-fold,while Pid1 and Angpt1 showed down-regulation over 50％.All observed expression changes demonstrated statistical significance(P＜0.01)through rigorous hypothesis testing.These findings highlight the potential involvement of these genes in thermogenic regulation,warranting further functional investigations to elucidate their molecular mechanisms in energy metabolism pathways.Conclusions This study established a novel"computational screening → in silico knockout → experimental validation"paradigm for tar-get discovery,systematically unveiling the molecular network involved in BAT thermogenic regulation.This methodology is equally applicable for identifying key regulatory genes in other physiological or pathological processes.The study identi-fied 11 core genes that may play pivotal regulatory roles during BAT thermogenic activation,which could potentially offer novel pharmacological intervention targets to improve energy metabolism and treat obesity-related complications.

Aim To systematically elucidate the molecular regulatory network of thermogenic function in brown adipose tissue(BAT)through multi-omics integrative analysis,to discover novel thermogenic regulatory genes and provide novel therapeutic targets for metabolic disorders.Methods A novel methodology for screening key genes regulating thermogenesis in BAT was constructed:First,differential expression analysis was performed on bulk RNA-seq data from murine BAT.Genes meeting the thresholds of ABS(log2FoldChange)＞1 and Padj＜0.05 were identified as differentially expressed genes.Intersectional analysis was then applied to obtain consensus upregulated and downregulated gene sets.Subsequently,scRNA-seq data of brown adipocytes were partitioned into high-expression group and low-expression group based on the expression levels of candidate genes.Differential analysis and gene set enrichment analysis(GSEA)were conducted between these groups to assess the correlation between candidate genes and thermogenic function.Finally,ex-perimental validation of selected candidate genes was performed using quantitative real-time PCR and Western blot.Results Bioinformatics analysis identified 65 thermogenesis-positive correlated genes and 7 thermogenesis-negative corre-lated genes.Subsequent quantitative PCR validation demonstrated that candidate genes Mfsd2a,Me1,Slc25a34,Pfkp,Ankrd9,Hsd17b12,Aldoa,Ctsz and Pcyt2 exhibited upregulation exceeding 5-fold,while Pid1 and Angpt1 showed down-regulation over 50％.All observed expression changes demonstrated statistical significance(P＜0.01)through rigorous hypothesis testing.These findings highlight the potential involvement of these genes in thermogenic regulation,warranting further functional investigations to elucidate their molecular mechanisms in energy metabolism pathways.Conclusions This study established a novel"computational screening → in silico knockout → experimental validation"paradigm for tar-get discovery,systematically unveiling the molecular network involved in BAT thermogenic regulation.This methodology is equally applicable for identifying key regulatory genes in other physiological or pathological processes.The study identi-fied 11 core genes that may play pivotal regulatory roles during BAT thermogenic activation,which could potentially offer novel pharmacological intervention targets to improve energy metabolism and treat obesity-related complications.

Objective: To investigate eye care behaviors among primary students in Wenzhou during Novel Coronavirus epidemic(COVID-19),and to provide a basis for eye care education and myopia prevention strategy. Methods: A total of 1 127 students from grade one to grade six of six primary schools in Wenzhou were selected to participate in an on-line invistigation regarding class attendance and eye care behaviors during the epidemic, March 6-9, 2020. Results: During the epidemic period, the primary school students in Wenzhou mainly took classes on the Internet (936, 83.1%), and the main learning tools were computers (391, 34.7%) and mobile phones (344, 30.5%), with an average of 3.00 h of online class. On average, students needed 2.00 h to complete homework and 1.00 h of extracurricular reading every day. In addition to learning, the daily use of mobile phone or ipad, computer and TV was 1.00,0.50,1.00 h respectively. Limited by the epidemic situation, the average daily exercise time of students was 0.81 h, including 0.00 h of outdoor activities; the average daily sleep was 9.00 h . During the epidemic period, 553 pupils (49.1%) reported eye discomfort, of which the most common was dry eyes (379, 47.4%). Multivariate Logistic regression analysis showed that the main learning tools of non-electronic products, reading distance > 30 cm or not reading, exercise time >0.5 h and outdoor activity time > 0.5 h were the protective factors of ocular discomfort (P<0.05). More than 2 h homework and recreational screen time higher than 0.5 h were risk factors for ocular discomfort(P＜0.05). Conclusion During the epidemic period, online learning increasedeye strain.Time of electronic devices usage,correct posture for reading,moderate level of ourdan physical activity,as well as prevention and treatment of eye strain should be strictly monitored.

Objective: To evaluate the aerosol concentration(PM2.5,PM10.0 and aerosol particle number) formation in non-contact "air-puff" tonometry and provide suggestions for medical workers to take appropriate daily protection during the prevalence of 2019-nCoV. Methods: A cross-sectional study was carried out in this study. Thirty healthy subjects were enrolled on February 22, 2020 at Eye Hospital of Wenzhou Medical University. The intraocular pressure (IOP) was measured by non-contact "air-puff" tonometer in the ophthalmic consulting room and the hall with or without masks. PM2.5, PM10.0 and aerosol particles were recorded by air quality detector. The cumulative effects of IOP measurement, PM2.5, PM10.0 and aerosol particle number were analyzed, and the aerosol density of subjects with and without masks was compared. Results: The PM2.5, PM10.0 and aerosol particles produced by the non-contact "air-puff" tonometry and increased with the increase of spray times. The IOP curves of 60 eyes of 30 subjects were measured respectively in two environments of medical consulting room and medical institution hall. It was found that PM2.5, pm10.0 and particle number fluctuated and increased with the increase of IOP measurement person times, showing cumulative effect, and the accumulation speed of aerosol density in hall was faster than that in consulting room. The density of PM2.5 and PM10.0 produced without gauze mask were (53.417±2.306) and (85.350± 3.488) μg/m³, which were higher than those of (50.567±0.862) and (80.617±1.463) μg/m³ with gauze mask. The differences were statistically significant (P=0.028, 0.019). Conclusions Aerosol can be produced by non-contact "air-puff" tonometer spraying, and it fluctuates with the increase of spraying times, showing a cumulative effect. The aerosol accumulation is higher in the hall with insufficient air circulation. And more aerosol can be produced without gauze mask.

The authors summarize Professor SHI Qi's clinical experience in diagnosing and treating chronic tendon and bone disease.The specific diagnosing and treating thinking and methods could be summarized as follows:1)Three stages,which means chronic tendon and bone disease could be treated according to early,medium and late stages.2) Three differentiations,which include differentiating disease,type and syndrome.3) Three examining,which include seeing patient clearly,reading the disease and getting the key point.In addition,Prof.SHI emphasizes threepoint syndrome differentiation which means the combination of the lesion's target,peri-target and whole syndrome characteristics differentiation.In the process of treatment,Prof.SHI emphasizes three methods combination of herb,technique and breathing technique.Both internal and external treatments should be used.Prof.SHI advocates that the control strategy should be the prevention,treatment and recuperation integration concept,including preventing disease,early treatment to prevent deterioration and preventing reoccurrence after cure.

Obj cetive A large number of recent studies show that sevoflurane anesthesia may cause learning and memory dysfunction.The aim of this study was to explore changes of learning and memory ability and hippocampal volume in infantile rats after neonatal interrupted and repeated inhalation of 2.6% sevoflurane through detecting the learning and memory ability by Morris water maze and the hippocampus volume by MRI.Method s Thirty two neonatal SD rats were randomly devided into two groups (n=16):experimental group and control group.Rats inhalated 2.6%sevoflurane in the experimental group and 1 L/min O2 +1 L/min Air in the control group at the postnatal days of 7, 14 and 21 (P7, P14, P21). The learning and memory ability was determined by the Morris water maze test from P31 to P37;The brains of rats were scanned by mag-netic resonance imaging ( MRI) machine under anesthesia with 1%sodium pentobarbital at P37, and the brain and bilateral hippocampal volumes were measured. Results ①In the place navigation test, the escape latency had no significant difference between the two groups (P>0.05).In the spatial probe test, the dwelling time, movement distance and number of entering times in platform quadrant decreased slightly in experimental group compared with those in the control group, while there was no significant difference (P>0.05).②The brain volume [(1.53 ±0.18) cm3 vs (1.60 ±0.13) cm3] and right hippocampal volume [(16.15 ±1.76)mm3 vs(16.46 ±1.71)mm3] had no significant difference between the two groups (P>0.05).The left hippocampal volume [(16.46 ±1.71)mm3] was decreased in the experimental group compared with the control group [(18.10 ±2.53)mm3](P<0.05). Conclusion The learning and memory ability has no significant changes in in-fantile rats after neonatal interrupted and repeated sevoflurane inhalation and MRI examination of hippocampal volume is not sufficient for the diagnosis of cognitive dysfunction.

Objective To evaluate the effects of multiple exposures to sevoflurane anesthesia on the expression of apolipoprotein E (ApoE) in the hippocampus of neonatal rats.Methods Twenty-four pathogen-free Sprague-Dawley rats (12 males,12 females),aged 7 days,weighing 15-20 g,were randomly divided into 2 groups (n=12 each) using a random number table:control group (group Con) and multiple exposures to sevoflurane anesthesia group (group Sev).On postnatal day 7,14 and 21,2.6％ sevoflurane was inhaled for 2 h in group Sev,while the mixed gas of oxygen and air was inhaled instead of sevoflurane in group Con.Morris water maze test was carried out on postnatal day 31-37 to assess cognitive function.The rats were then sacrificed,and the hippocampus was removed to determine the expression of ApoE protein in hippocampal CA1,CA3 and DG regions (by immunohistochemistry) and the expression of ApoE mRNA (by fluorescent quantitative real-time reverse transcriptase polymerase chain reaction).Results There was no significant difference between the two groups in cognitive function (P＞0.05).Compared with group Con,the expression of ApoE protein in hippocampal CA1 and CA3 regions and ApoE mRNA was significantly up-regulated in group Sev (P＜0.05),and no statistically significant change was found in the expression of ApoE protein in hippocampal DG region in group Sev (P＞0.05).Conclusion Multipie exposures to sevoflurane anesthesia can up-regulate the expression of hippocampal ApoE and produce mild neurotoxicity without causing changes in cognitive function in neonatal rats.