ObjectiveBased on transcriptomics， to explore the mechanism of Hei Xiaoyaosan regulating the phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway to improve the learning and memory ability of insomnia rats with liver depression syndrome. MethodsSixty 8-week-old male SD rats were randomly divided into the blank group， model group， eszopiclone group （0.09 mg·kg^-1）， and low， medium， and high dose groups of Hei Xiaoyaosan （3.82， 7.65， 15.30 g·kg^-1）， with ten rats in each group. Except for the blank group， the other groups were induced insomnia rat model with liver depression by chronic restraint， tail clamping stimulation and intraperitoneal injection of p-chlorophenylalanine （PCPA）. Each treatment group received intragastric administration according to the specified dosage， once a day for 14 consecutive days. The pentobarbital sodium cooperative sleep test， open field test， and Morris water maze test were used to test the sleep quality， depressive-like behavior， and learning and memory abilities of rats. Additionally， enzyme-linked immunosorbent assay （ELISA） was used to detect the contents of 5-hydroxytryptamine （5-HT）， γ-aminobutyric acid （GABA）， brain-derived neurotrophic factor （BDNF）， glial cell line-derived neurotrophic factor （GDNF） and nitric oxide （NO） in hippocampus. Hematoxylin-eosin （HE） staining was performed to observe pathological changes of the hippocampal tissue， while terminal deoxynucleotidyl transferase deoxyuridine triphosphate （dUTP） nick end labeling （TUNEL） was used to evaluate apoptosis of hippocampal neurons. Transcriptomic sequencing technology was employed to identify differentially expressed genes in hippocampus between the model group and the blank group， as well as between the medium-dose group of Hei Xiaoyaosan and the model group. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis were performed on the intersecting genes. Subsequently， the enriched key genes and signaling pathways were analyzed and verified. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was utilized to assess the mRNA expression levels of phosphatase and tensin homolog （PTEN）， B-cell lymphoma-2 （Bcl-2）-like protein 11 （BCL2L11）， and mitogen-activated protein kinase 1 （MAPK1） in hippocampus， and Western blot was employed to evaluate the protein expressions of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， Bcl-2， Bcl-2-associated X protein （Bax）， and cleaved Caspase-3 in the same tissue. ResultsCompared with the blank group， the model group exhibited a reduction in body weight， an increase in sleep latency， and a decrease in sleep duration （P<0.01）. Additionally， rats showed obvious depression-like behavior， and their learning and memory abilities decreased. Furthermore， the contents of 5-HT， GABA， NO， BDNF and GDNF in hippocampus decreased （P<0.01）. Histological examination revealed a disorganized cell arrangement in the CA1 region of the hippocampus， characterized by irregular cell shapes， a reduced cell count， deeply stained and pyknotic nuclei， increased vacuolar degeneration， and an elevated apoptosis rate （P<0.01）. Compared with the model group， the body weight of the high and medium dose groups of Hei Xiaoyaosan increased， the sleep latency shortened and the sleep time prolonged （P<0.05， P<0.01）. Additionally， depression-like behavior and learning and memory abilities of rats were significantly improved， the levels of 5-HT， GABA， NO， BDNF and GDNF in the hippocampus increased （P<0.05， P<0.01）. These interventions also ameliorated pathological damage in the hippocampal CA1 area and reduced the apoptosis of hippocampal neurons （P<0.01）. Transcriptomic sequencing results indicated that Hei Xiaoyaosan might exert a therapeutic effect by regulating PI3K/Akt pathway through key mRNAs such as PTEN， BCL2L11， and MAPK1. The roles of these key mRNAs and proteins within PI3K/Akt pathway were further validated. In comparison to the blank group， the expression levels of PTEN， BCL2L11 and MAPK1 mRNA in the hippocampus of rats in the model group were increased （P<0.01）， while the protein expression levels of p-PI3K， p-Akt and Bcl-2 were decreased （P<0.01）， and the protein expression levels of PTEN， Bax and cleaved Caspase-3 were increased （P<0.01）. Compared with the model group， the high-dose and medium-dose groups of Hei Xiaoyaosan could down-regulate the expressions of PTEN， BCL2L11 and MAPK1 mRNAs （P<0.01）， up-regulate the expressions of p-PI3K， p-Akt and Bcl-2 proteins （P<0.01）， and down-regulate the protein expressions of PTEN， Bax and cleaved Caspase-3 （P<0.05， P<0.01）. ConclusionHei Xiaoyaosan may regulate PI3K/Akt signaling pathway by down-regulating expressions of key genes such as PTEN， BCL2L11 and MAPK1， and thus improve the learning and memory abilities of insomnia rats with liver depression syndrome.

ObjectiveTo understand the visual environment sanitation in primary and secondary school classrooms in Minhang District， Shanghai， and to investigate the factors affecting the decline in students’ visual acuity. MethodsIn 2020， among all the primary and secondary schools in Minhang District， according to different types of classroom structures， levels， orientations， unilateral lighting， and bilateral lighting， a total of 20 231 students from 674 classrooms in primary and secondary schools were selected through stratified cluster sampling. Each monitored classroom was regarded as a research unit， and the naked eye vision of students was tested using a standard logarithmic vision chart during the 2020 and 2021 academic years. The change in average visual acuity between the two measurements was taken as the dependent variable， and the visual environment indices were considered as independent variables. The relationship between classroom visual environment indices and the decline in students’ naked eye vision was analyzed. ResultsThe qualification rates for per capita classroom area， window to floor area ratio， average illuminance of the blackboard， uniformity of blackboard illuminance， average illuminance of the desks， uniformity of desk illuminance， lighting coefficient of classroom， blackboard reflection ratio， back wall reflection ratio， and distance between lamps and desks were 79.53%， 88.58%， 46.74%， 70.33%，64.69%， 80.86%， 71.81%， 20.30%， 1.63%， and 97.53%， respectively. The average naked eye vision of primary and secondary school students in the 2020 academic year was 4.75±0.37 （right eye） and 4.76±0.37 （left eye）， while in the 2021 academic year， it was 4.70±0.39 （right eye） and 4.71±0.38 （left eye）. There was a significant decrease in the naked eye vision of primary and secondary school students in the 2021 academic year compared to that of 2020 （P<0.05）. Univariate linear regression analysis showed a significant correlation between grade， average illuminance of the blackboard， average illuminance of the desks， uniformity of desk illuminance， lighting coefficient， and distance between lamps and desks， and the decline of students’ naked eye vision within one academic year （P<0.05）. After adjusting for grade， multiple linear regression analysis revealed a significant correlation between the average illuminance of the blackboard， uniformity of desk illuminance， lighting coefficient， and distance between lamps and desks， and the decline of students’ naked eye vision within one academic year （P<0.05）. Higher average illuminance of the blackboard， uniformity of desk illuminance， lighting coefficient， and distance between lamps and desks were protective factors for naked eye vision. ConclusionThe visual environment of primary and secondary school classrooms in Minhang District is a matter of concern. Average illuminance of the blackboard， uniformity of desk illuminance， lighting coefficient， and distance between lamps and desks are related to the degree of visual impairment in students over a year.

ObjectiveTo investigate the general situation of defect of vertebral column among primary and middle school students in Minhang District of Shanghai and analyze the related factors， to provide a scientific basis for prevention and treatment. MethodsFrom September to October 2022， a total of 5 715 students were selected from two primary schools， two middle schools， and two high schools in Minhang District for physical examinations and screening for defect of vertebral column. ResultsTotally 219 students had defect of vertebral column， accounting for 3.83% of the sampled population. Anteroposterior spinal abnormalities were found in 4 individuals， accounting for 0.07%， and 218 students had scoliosis， accounting for 3.81%. The detection rate of defect of vertebral column was higher in girls （6.27%） than that in boys （1.51%）， and higher in high school students （10.74%） than in primary school students （1.31%） and middle school students （10.97%）. Students who are mildly underweight （5.95%） and who are moderately to severely underweight （7.46%） had a higher detection rate of defect of vertebral column than those with normal weight （4.54%）， overweight （2.83%）， and obesity （1.60%）. The detection rate among students with poor vision （4.32%） was significantly higher than those with normal vision （2.24%）， with all differences statistically significant （all P<0.05）. ConclusionThe positive rate of defect of vertebral column in primary and middle school students in Minhang District， Shanghai is nearly 4%， with most cases being scoliosis. Factors such as being female， increasing age， being underweight， and poor vision are associated with a higher probability of detecting defect of vertebral column.

Objective To investigate the effect of plasma microRNA(miR)-93-5p on ovarian granulosa cell proliferation in patients with polycystic ovary syndrome(PCOS)and its possible mechanism.Methods A total of 120 PCOS patients of childbearing period who were treated at Jiaozuo People's Hospital from January 2022 to January 2023 were selected as the PCOS group,and 18 healthy women of childbearing period who were physically examined at the same hospital during the same period were selected as the control group.The age,body weight and height of the subjects in the two groups were recorded,and the body mass index(BMI)was calculated.Fasting venous blood was collected from the subjects during the follicular phase of the natural menstrual cycle or the progesterone-induced withdrawal bleeding phase,and serum levels of luteinizing hormone(LH),follicle-stimulating hormone(FSH),total testosterone(TT),anti-Mullerian hormone(AMH),aspartate aminotransferase(AST),alanine aminotransferase(ALT),fasting insulin(FINS),and fasting blood glucose(FBG)were measured by using the electrochemical method;and the homeostasis model assessment of insulin resistance(HOMA-IR)was calculated.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression level of miR-93-5p in plasma of patients in the two groups.Human ovarian granulosa cells(KGN cells)in logarithmic growth phase were cultured in 6-well plates with 3 × 105 cells per well,and they were randomly divided into the miR-93-5p mimics group,LY294002+miR-93-5p group,AZD5363+miR-93-5p group,negative control(NC)group,and blank control group.The KGN cells in the miR-93-5p group were transfected with miR-93-5p mimics;the KGN cells in the LY294002+miR-93-5p group were transfected with miR-93-5p mimics and treated with 50 mmol·L-1 LY294002 one hour before transfection;the KGN cells in the AZD5363+miR-93-5p group were transfected with miR-93-5p mimics and treated with 50 μmol·L-1 AZD5363 one hour before transfection;the KGN cells in the NC group were transfected with the negative control plasmids;and the KGN cells in the blank control group were not treated at all.The expression of miR-93-5p in the KGN cells in each group was detected by qRT-PCR,and the proliferation of the KGN cells in each group was detected by cell counting kit-8.Results There was no significant differences in age,FSH,ALT,and AST levels of patients between the PCOS group and the blank control group(P＞0.05).The BMI,TT,AMH,LH,FINS,FBG,and HOMA-IR of patients in the PCOS group were significantly higher than those in the blank control group(P＜0.05).The relative expression of miR-93-5p in plasma of patients in the PCOS group was significantly higher than that in the blank control group(t=-5.549,P＜0.001).miR-93-5p was moderately positively correlated with TT,FINS and HOMA-IR(r=0.434,0.622,0.586;P＜0.001)and was mildly positively correlated with FBG and LH(r=0.398,0.398;P＜0.001).The receiver operating characteristic curve showed that the optimal cut-off value for plasma miR-93-5p in diagnosing PCOS was 1.380,the area under the curve was 0.906(95％confidence interval:0.839-0.973,P＜0.001),the sensitivity was 0.858,the specificity was 0.833,and the Youden index was 0.691.The relative expression of miR-93-5p in the KGN cells in the miR-93-5p mimics group,LY294002+miR-93-5p group and AZD5363+miR-93-5p group was significantly higher than that in the blank control group and NC group(P＜0.05).After 24,48 and 72 hours of culture,the proliferation of the KGN cells in the miR-93-5p mimics group,LY294002+miR-93-5p group and AZD5363+miR-93-5p group was significantly higher than that in the blank control group and the NC group(P＜0.05);the proliferation of the KGN cells in the LY294002+miR-93-5p group and AZD5363+miR-93-5p group was significantly lower than that in the miR-93-5p mimics group(P＜0.05).Conclusion miR-93-5p in plasma is overexpressed in PCOS patients,and it may be involved in the occurrence and development of PCOS by mediating the proliferation of ovarian granulosa cells through the phosphoinositide 3 kinase/protein kinase B signaling pathway.The miR-93-5p level in plasma has a certain diagnostic value for PCOS.

Objective To explore the expression level of microRNA(miR)-320a-3p in peripheral blood in patients with polycystic ovary syndrome(PCOS)and its possible mechanism for regulating PCOS.Methods A total of 149 PCOS patients admitted to the Jiaozuo People's Hospital from July 2021 to July 2022 were selected as the research subjects(PCOS group),and 18 healthy volunteers with a regular menstrual cycle(28-35 d),no clinical or biochemical manifestations of hyperandrogenism,and no polycystic ovary changes detected by ultrasonography were selected as the control group.Clinical data of the subjects in the two groups were collected and compared.The expression level of miR-320a-3p in plasma of subjects in the two groups was detected by using the real-time quantitative polymerase chain reaction.The correlation between plasma miR-320a-3p expression and clinical indexes was evaluated by using the Pearson partial correlation analysis.The predictive value of miR-320a-3p for PCOS was analyzed by using the receiver operating characteristic curve.The relationship between miR-320a-3p and androgen receptor was evaluated by using the dual luciferase reporter gene assay.Results There was no significant difference in age,hip circumference,follicle stimulating hormone(FSH),prolactin(PRL),and high-density lipoprotein cholesterol(HDL-C)between the two groups(P＞0.05).The body mass index,waist circumference,total testosterone(TT),anti-Miillerian hormone(AMH),luteinizing hormone(LH),fasting insulin(FINS),fasting blood glucose(FBG),2-hour postprandial blood glucose(2 h BG),homeostasis model assessment of insulin resistance(HOMA-IR),total cholesterol,triglycerides(TG),and low-density lipoprotein cholesterol(LDL-C)of patients in the PCOS group were significantly higher than those in the control group,while the level of estradiol(E2)was significantly lower than that in the control group(P＜0.05).The relative expression level of miR-320a-3p in plasma of patients in the PCOS group was significantly lower than that in the control group(P＜0.05).The expression level of miR-320a-3p was moderately negatively correlated with TT(r=-0.594,P＜0.05)and slightly negatively correlated with waist circumference,FINS,2 h BG,HOMA-IR,and LDL-C(r=-0.293,-0.208,-0.227,-0.208,-0.208;P＜0.05),and showed no significant correlation with hip circumference,AMH,FSH,E2,PRL,FBG,TG,and HDL-C(r=-0.079,0.020,-0.042,0.089,0.005,-0.141,-0.116,0.059;P＞0.05).The area under the curve of miR-320a-3p for predicting PCOS was 0.968(95％confidence interval:0.922-1.000,P＜0.05),with a cut-off value of 0.515,a sensitivity of 0.917,a specificity of 0.789,and a Jordon index of 0.706.miR-320a-3p negatively regulated androgen receptors.Conclusion miR-320a-3p is abnormally low expressed in peripheral blood of PCOS patients and may participate in the occurrence and development of PCOS by negatively regulating androgen receptors to increase TT levels.It has high predictive value for diagnosing PCOS.

Sex difference has shown in the arthritis diseases in human population and animal models. We investigate how the sex and symmetry vary among mouse models with different genomic backgrounds. Disease data of sex and limbs accumulated in the past more than two decades from four unique populations of murine arthritis models were analyzed. They are (1) interleukin-1 receptor antagonist (IL-1ra) deficient mice under Balb/c background (Balb/c KO); (2) Mice with collagen II induced arthritis under DBA/1 background; (3) Mice with collagen II induced arthritis under C57BL/6 (B6) background and (4) A F2 generation population created by Balb/c KO X DBA/1 KO.Our data shows that there is a great variation in sexual dimorphism for arthritis incidence and severity of arthritis in mice harboring specific genetic modifications. For a F2 population, the incidence of arthritis was 57.1% in female mice and 75.6% in male mice. There was a difference in severity related to sex in two populations: B6.DR1/ B6.DR4 (P < 0.001) and F2 (P = 0.023) There was no difference Balb/c parental strain or in collagen-induced arthritis (CIA) in DBA/1 mice. Among these populations, the right hindlimbs are significantly higher than the scores for the left hindlimbs in males (P < 0.05). However, when examining disease expression using the collagen induced arthritis model with DBA/1 mice, sex-dimorphism did not reach statistical significance, while left hindlimbs showed a tendency toward greater disease expression over the right. Sexual dimorphism in disease expression in mouse models is strain and genomic background dependent. It sets an alarm that potential variation in sexual dimorphism among different racial and ethnic groups in human populations may exist. It is important to not only include both sexes and but also pay attention to possible variations caused by disease expression and response to treatment in all the studies of arthritis in animal models and human populations.

【Objective】 To explore the characteristics of esophageal motility and reflux of endoscopic-negative heartburn patients based on the Lyon Consensus Diagnostic Criteria and discuss the differential diagnosis value of the mean nocturnal baseline impedance （MNBI） and the postreflux swallow-induced peristaltic wave （PSPW） index for reflux hypersensitivity （RH） and functional heartburn （FH） patients. 【Methods】 We enrolled 132 patients with heartburn as the main symptom who visited the Gastroenterology Department of our hospital from January 2017 to June 2021， including 24 in the non-erosive reflux disease （NERD） group， 24 in the RH group， and 84 in the FH group. All the patients completed gastroscopy， esophageal high-resolution manometry and 24h-pH impedance monitoring. We analyzed and compared the related indexes of esophageal motility and reflux. The receiver operating characteristic （ROC） curve was used to analyze the esophageal proximal MNBI， distal MNBI， and PSPW indexes for the differential diagnosis of RH and FH. 【Results】 The lower esophageal sphincter （LES） average resting pressure， LES residual pressure and intact relaxation pressure （IRP） in NERD were lower than those in RH and FH （P<0.05）. The number of acid reflux， the upright acid exposure time （AET）， the supine position AET， the total AET， and the DeMeester score were higher in NERD than in RH and FH （P<0.05）. The non-acid reflux， distal MNBI and PSPW indexes were lower in NERD than in RH and FH （P<0.05）. The distal MNBI and PSPW indexes were lower in RH than in FH （P<0.05）. However， there was no significant difference in proximal MNBI among the three groups （P>0.05）. When the proximal MNBI， distal MNBI， and PSPW indexes were used alone respectively to diagnose RH， the area under the ROC curve was 0.480， 0.810， 0.682， respectively， with the sensitivities being 87.5%， 100% and 91.7%， and the specificities being 26.2%， 66.7% and 51.2%， respectively. 【Conclusion】 NERD patients have obvious abnormal esophageal dynamics， mainly manifested as LES relaxation， which further aggravates the pathological acid reflux， while patients with RH and FH are mainly exposed to physiological acid or non-acid reflux. In patients with endoscopic negative heartburn， distal MNBI value can improve the clinical diagnosis rate of RH and help distinguish RH from FH.

Objective To investigate the resting brain functional connectivity in patients with post-stroke cognitive impairment (PSCI).Methods From January, 2020 to January, 2021, 24 PSCI patients were divided into mild group (n=12) and moderate group (n=12) according to Mini-Mental State Examination and Montreal Cognitive Assessment scores; and other ten healthy controls (control group) were included. All the subjects were scanned with 64-channel functional near-infrared spectroscopy in resting for six minutes. The data were analyzed with NirSpark 1.6.12 and SPSS 26.0.Results Using deoxyhemoglobin, the functional connectivity between bilateral hemispheres reduced in the moderate group compared with those in the control group (t=-2.763, P=0.024), especially for the functional connectivity between sensorimotor cortexes (F=12.674, P=0.031).Conclusion Functional connectivity between hemispheres decreases in PSCI patients, especially between sensorimotor cortexes.

Objective:To investigate the prevalence and independent risk factors of non-alcoholic fatty liver disease (NAFLD) and advanced chronic liver disease among the type 2 diabetes mellitus (T2DM) population in the Shenyang community, so as to provide evidence for the prevention and control of T2DM combined with NAFLD.Methods:This cross-sectional study was conducted in July 2021. 644 T2DM cases from 13 communities in Heping District, Shenyang City were selected. All the surveyed subjects underwent physical examination (measurements of height, body mass index, neck circumference, waist circumference, abdominal circumference, hip circumference, and blood pressure), infection screening (excluding hepatitis B and C, AIDS, and syphilis), random fingertip blood glucose, controlled attenuation parameter (CAP), and liver stiffness measurement (LSM). The study subjects were divided into the non-advanced chronic liver disease group and the advanced chronic liver disease group according to whether the LSM value was greater than 10 kPa. Cirrhotic portal hypertension development was indicated in patients with LSM ≥ 15 kPa. The comparison of multiple mean values among the sample groups was performed by analysis of variance when the normal distribution was met.Results:In the T2DM community population, there were 401 cases (62.27%) combined with NAFLD, 63 cases (9.78%) combined with advanced chronic liver disease, and 14 cases (2.17%) combined with portal hypertension. There were 581 cases in the non-advanced chronic liver disease group and 63 cases (9.78%) in the advanced chronic liver disease group (LSM ≥10 kPa), including 49 cases (7.61%) with 10 kPa≤LSM<15 kPa, 11 cases (1.71%) with 15 kPa ≤LSM<25 kPa, and 3 cases (0.47%) with LSM ≥ 25 kPa. Age, body mass, body mass index, neck circumference, waist circumference, hip circumference, waist-to-height ratio, systolic blood pressure, and CAP were all statistically different between the non-advanced chronic liver disease group and the advanced chronic liver disease group ( F=-1.983,-2.598,-4.091,-2.062,-3.909, -4.581,-4.295,-2.474, and -5.191, respectively; P<0.05). There was a statistically significant difference in terms of whether or not there was combined cerebrovascular disease (2=4.632, P=0.031); however, there were no statistically significant differences in terms of lifestyle, diabetes complications, and other complications ( P>0.05). Conclusion:Patients with T2DM have a higher prevalence of NAFLD (62.27%) than those with advanced chronic liver disease (9.78%). 2.17% of T2DM cases in the community may not have had early diagnosis and early intervention, and they might have been combined with cirrhotic portal hypertension. So, the management of these patients should be strengthened.

Directed evolution is a cyclic process that alternates between constructing different genes and screening functional gene variants. It has been widely used in optimization and analysis of DNA sequence, gene function and protein structure. It includes random gene libraries construction, gene expression in suitable hosts and mutant libraries screening. The key to construct gene library is the storage capacity and mutation diversity, to screen is high sensitivity and high throughput. This review discusses the latest advances in directed evolution. These new technologies greatly accelerate and simplify the traditional directional evolution process and promote the development of directed evolution.
Base Sequence ; Directed Molecular Evolution ; Gene Library ; Mutation ; Proteins/genetics*