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25 September 2021, Volume 42 Issue 9

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Basic Researches

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Prokaryotic expression of enterovirus 71 type VP1 protein and its application in high throughput antibody screening

Ke Cui, Yaru Kong, Chuanmin Ma, Zhenjie Zhang, Weifeng Shi, Chuansong Quan

2021, 42(9):  641-644.  DOI: 10.3969/j.issn.2097-0005.2021.09.001

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Enterovirus 71 （EV71） is the main pathogen causing severe hand， foot and mouth disease （HFMD）， however， there is no effective antiviral drugs available. To screen mouse specific B cells expressing antibodies against EV71 by using EV71 VP1 protein as an antigen.

The codon-optimized EV71 VP1 gene was cloned into pET-21a （+） expression vector and transformed into E. coli. BL21-DE3. By optimizing protein expression and renaturation conditions， the expressed protein was confirmed by SDS-PAGE and Western blot and its antigenicity was also tested. Using purified protein as antigen， B lymphocytes of mice immunized with inactivated， and EV71 viruses were sorted by flow cytometry for the preparation of monoclonal antibody.

The EV71 VP1 gene was stably expressed in the form of inclusion body in the E. coli. The purified protein showed good immunogenicity， and there was a significant difference between the B cells of immunized and unimmunized BALB/c mice when screened using the expressed protein.

The recombinant EV71 VP1 protein with good immunogenicity is successfully obtained and combined with flow cytometry， it can be used for detection and sorting of EV71 specific B cells.

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Attenuation of neurological deficit after SAH and the promotion of microglia polarization toward M2 phenotype in mice by the novel Nrf2 activator CDDO-EA

Qiwei Dong, Huayang Zhao, Shihao Ren, Pengjie Zhang, Yajun Hou, Leilei Mao

2021, 42(9):  645-650.  DOI: 10.3969/j.issn.2097-0005.2021.09.002

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To investigate the effects of CDDO-EA （CDDO-ethylamide）， a novel Nrf2 related factor 2 （Nrf2）?， on neurological deficit and microglia polarization after subarachnoid hemorrhage （SAH） in mice.

Thirty healthy C57BL/6 mice were randomly divided into sham group， SAH + PBS group and SAH + CDDO-EA （50 μg） group. The SAH model was made by middle cerebral artery puncture， and the mice were sacrificed 3 days after operation for neurological function score. Brain tissue was collected for frozen sections and the M2-type microglia cells were labeled with CD206/Iba1 by immunofluorescence double staining.

The cerebral cortex of each group was collected 48 h after operation to detect the expression of CDDO-EA by Western blot. Compared with PBS control group， the expression of Nrf2 in brain of 50 μg and 100 μg treatment groups was significantly increased （P < 0.05）.The mice received PBS or CDDO-EA（50 μg） after surgery by intraperitoneal injection. The neurological function score was evaluated 72 h after surgery. SAH mice had obvious neurological dysfunction， but the neurological function was improved after CDDO-EA treatment （P < 0.05）. In addition， the numberof M2 microglia in cerebral cortex and hippocampus after CDDO-EA treatment significantly increased than that in vehicle control group.

CDDO-EA treatment attenuates neurological deficits after SAH， which may be achieved by up-regulating the expression of Nrf2 and promoting microglia polarization toward M2 phenotype.

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Interference with UCP2 in protection of H9C2 cardiomyocytes from hypoxia-induced apoptosis and inhibition of autophagy

Li Zhang, Feng Li, Yueyan Liu, Yanmei Wang, Chenchen Zhang

2021, 42(9):  651-656.  DOI: 10.3969/j.issn.2097-0005.2021.09.003

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To study the effect of interfering uncoupling protein 2 expression on apoptosis and autophagy induced by hypoxia.

The expressions of UCP2 in H9C2 myocardial cells after hypoxia were detected by RT-q PCR. Reactive oxygen species （ROS） fluorescence assay was used to detect the effect of UCP2 interference on intracellular ROS level. The apoptotic rate was detected by flow cytometry. The effects of UCP2 interference on cleaved-caspase 3 were further detected by Western blotting. The cell damage related factors were detected by ELISA： lactate dehydrogenase （LDH）， creatine kinase （CK）， interleukin-6 （IL-6） and tumor necrosis factor-alpha （TNF-a）. The expression levels of membrane LC3/cytoplasmic LC3 （LC3-?Ⅱ/LC3-?Ⅰ）， autophagy gene Beclin-1 and p62 were detected.

The expression of UCP2 was significantly increased in H9C2 cells under hypoxia. Interference with UCP2 expression could reduce the increase of ROS level in H9C2 cells induced by hypoxia. Interference with UCP2 expression could reduce the apoptotic rate of H9C2 cells and the expression levels of LDH， CK， IL-6 and TNF-alpha in hypoxic environment. In Western blotting， interfering with UCP2 expression could decrease the expression of LC3-?Ⅱ/LC3-?Ⅰ and Beclin-1， and increase the expression of p62.

Interference with UCP2 can reduce ROS level in cells induced by hypoxia， and inhibit apoptosis and autophagy induced by hypoxia.

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Influences of Chinese medicinal with actions of invigorating blood circulation， softing scleroma and firming collaterals prescription on coronary microvascular thrombosis and endothelial injury factors in rats

Meng Zhang, Ming Li, Li Zhang, Ming Zhang, Xin Miao, Yuanmin Li

2021, 42(9):  657-661.  DOI: 10.3969/j.issn.2097-0005.2021.09.004

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To investigate the effect of Chinese medicinal with actions of Invigorating blood circulation， softing scleroma and firming collaterals prescription on coronary microvascular thrombosis and endothelial injury factor in rats.

60 Wistar rats were randomLy divided into： Sham operation group， Model group， Nicorandil group and Chinese medicine group.The sham operation group was used as the control group.The model was established by injecting sodium laurate into the left ventricle of rats to establish rat models of coronary microvascular hrombosis and endothelial function injury.The drug group was given Nicorandil 1.5 mg·（kg·d）^－1， Traditional Chinese Medicine prescription 15 g·（kg·d）^－1， Sham operation group and Model group were given the same amount of distilled water. After the 28th day，treatment was selected as the observation point to observe the myocardial pathological changes and coronary microvascular thrombosis occlusion， and to detect the serum levels of Prostaglandin Ⅰ₂（PGI₂） and Thromboxane A₂（TXA₂）.

Compared with the sham operation group， the left ventricular injection of sodium laurate in the model group led to coronary microvascular thrombosis and myocardial tissue pathological damage； After 28 days of medication， compared with the model group， the traditional Chinese medicine group could reduce microvascular embolism， improve myocardial pathological damage， increase serum prostaglandin Ⅰ₂ （PGI₂） and reduce thromboxane A₂ （TXA₂）； Compared with the nicorandil group， there was no significant difference in the improvement of microvascular thrombosis and the reduction of endothelial injury factor in the traditional Chinese medicine group.

Sodium laurate can cause coronary microvascular thrombosis and endothelial injury in rats.For 28 days， the traditional Chinese medicine group could reduce the pathological state of coronary microvascular embolization and improve the endothelial function of microvascular lesions.Compared with Nicordil， there was no obvious advantage in Chinese medicine group.

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Alleviation effects of drinking hydrogen-rich water on the oral mucosal ulcer induced by 5-fluorouracil in rats

Danya Zhang, Junli Xue, Boyan Liu, Geru Tao, Yunzhi Shi, Shucun Qin

2021, 42(9):  662-666.  DOI: 10.3969/j.issn.2097-0005.2021.09.005

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To investigate the alleviation effects of drinking hydrogen-rich water on the oral mucosal ulcer induced by 5-fluorouracil （5-FU） and its possible biological mechanism.

Male SD rats were intraperitoneally injected with 5-FU （0.05 mg/g） for 2 consecutive days. On D3， rats were treated with NaOH particles or 20% acetic acid on buccal mucosa to establish oral ulcer model. From D5， the rats were given hydrogen-rich water， and the animals were sacrificed on the D10. The survival rates of rats in all experimental groups were counted， and the lesions of oral mucosa were measured by Image J software. The levels of TNF-α， IL-6 and IL-1β in serum were detected by ELISA， and the expressions of TNF-α， IL-6 and iNOS at buccal mucosa were detected by immunohistochemistry.

Drinking hydrogen-rich water improved the survival rate of rats in the experimental groups. The damage area of ulcers produced by NaOH particles were larger than that caused by injection with 20% acetic acid （P < 0.05）， but there was no significant difference in the damage area of ulcers with drinking hydrogen-rich water or not （P > 0.05）. The serum levels of TNF-α， IL-6 and IL-1β of rats in model groups were significantly higher than those in the control group， while levels of the hydrogen-rich water group showed a downward trend compared with the model group. The expression of TNF-α and IL-6 at buccal mucosa were low in the healthy control and high in the model groups， while the expression in hydrogen-rich water groups showed a downward trend compared with the model group. The expression of iNOS at buccal mucosa was low in all groups.

Molecular hydrogen alleviates oral mucosal ulcers induced by 5-FU in rats possibly through inhibiting oxidative stress-related inflammation.

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Study of survivin expression and angiogenesis in oral squamous cell carcinoma

Tengyu Li, Xia Zhang, Xiaopeng Yu, Xiaoguang Li, Xiran Wang, yanxiu Wang, Huanyan Liu

2021, 42(9):  667-669.  DOI: 10.3969/j.issn.2097-0005.2021.09.006

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To explore the relationships between Survivin expression and angiogenesis in oral squamous cell carcinoma.

The Survivin， COX-2， VEGF and CD34 expressions were evalulated by immunohistochemical method.

The expressions of Survivin and COX-2， VEGF in oral squamous cell carcinoms were significantly positively correlated（P < 0.05）. There was significant difference of MVD between Survivin positive oral squamous cell carcinoma and negative ones（P < 0.01）.

The present findings suggest that there is some link between Survivin and angiogenesis in oral squamous cell carcinom， partly attributing to the effect of COX-2 and VEGF.

Clinical Researches

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The effect of DEHSI and PVL on brain development maturity in preterm infants

Yaodan Ban, Meng Xu, Jing Jin, Deting Ma

2021, 42(9):  670-674.  DOI: 10.3969/j.issn.2097-0005.2021.09.007

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To evaluate the effects of diffuse excessive high signal intensity （DEHSI） and periventricular leukomalacia （PVL） on brain development maturity of premature infants by total maturational scores （TMS）.

Retrospective analysis was performed on the craniocerebral MR images of 68 neonates， which were divided into three groups： normal preterm infants group， DEHSI group and PVL group （adjusted gestational age： 37－44 weeks）. The craniocerebral MR images of each newborn were evaluated by the TMS. The TMS scores were compared between the groups.

There were statistically significant differences in myelination， cortical infolding and TMS scores between the normal preterm infants and the DEHSI preterm infants （P < 0.05）；In the scores of myelination， cortical infolding and TMS， the order from high to low was normal preterm infants group， PVL infants group， DEHSI preterm infants group；There were no statistically significant differences in germinal matrix and migrating glial cells scores between any two groups （P > 0.05）；

The presence of DEHSI and PVL in the white matter of preterm infants affects the developmental maturity of the brain， and DEHSI and PVL have different effects on the developmental maturity of the brain. DEHSI does delay the developmental maturity of the brain.

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The diagnostic value of peripheral blood NLR， PLR and SII in thyroid nodules

Zhongwei Fang, Xianwei Zhou, Zhiqiang Cao, Yanfang Song, Gengpu Ren

2021, 42(9):  675-679.  DOI: 10.3969/j.issn.2097-0005.2021.09.008

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To investigate the values of the differential diagnosis of peripheral blood neutrophil/lymphocyte ratio （NLR）， platelet/lymphocyte ratio （PLR） and systemic immune inflammation index （SII） in healthy people， benign and malignant thyroid nodules.

172 patients with benign thyroid nodules and 285 patients with malignant nodules diagnosed in the Second People's Hospital of Liaocheng from January 2017 to October 2019 were selected as the research objects， and 199 patients with health examinations in the hospital during the same period were selected as the control group， the preoperative peripheral blood neutrophil count， lymphocyte count and platelet count were recorded， and NLR， PLR and SII were calculated， and NLR， PLR and SII in healthy people， benign nodules， and malignant nodules were compared by t test for the difference between groups， the receiver operating characteristic curve （ROC curve） was used to determine the critical values of NLR， PLR， and SII in different groups.

Except that there was no statistically significant difference between NLR and SII in the comparison of benign and malignant nodules （group A vs group B）， the three indicators （NLR， PLR， SII） in the other three groups were compared with each other， and the differences were statistically significant （P < 0.05）. The area of AUC was between 0.72 and 0.8. The sensitivity of NLR， PLR， and SII in the identification of healthy people and benign thyroid nodules was 85.9%， 92.5%， and 81.9% respectively. In the detection of healthy people and malignant thyroid nodules， the sensitivity in the discrimination was 70.4%， 81.9%， and 74.1%， respectively. The discrimination specificities in the benign nodule group and the malignant nodule group were 85.6%， 69.5%， and 83.2%， respectively， and the significance was significant.

The detection of NLR， PLR， and SII has a certain value to distinguish between healthy people and benign nodules and healthy people and thyroid nodules， but it is not significant to distinguish between benign and malignant nodules.

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Discussion on the reference range of blood lipids during pregnancy in Wujiang area of Suzhou city

Xu Zhang, HongLin Yang

2021, 42(9):  680-684.  DOI: 10.3969/j.issn.2097-0005.2021.09.009

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To observe the distribution of blood lipids during different gestation periods in Wujiang District of Suzhou City， to establish the reference range of this region， and to provide reference for clinicians to make decisions.

A total of 1388 pregnant women with single pregnancy who were registered in our hospital until their delivery to full term were selected as the research subjects. The blood triglyceride， total cholesterol， low density lipoprotein and high density lipoprotein of 11 to 13⁺⁶ weeks of pregnancy， 28 to 31⁺⁶ weeks of pregnancy， 35 to 36⁺⁶ weeks of pregnancy and 38 to 41⁺⁶ weeks of pregnancy were analyzed statistically to establish the reference range of blood lipids of pregnant women at various stages of pregnancy in the local area.

The level of blood lipid during pregnancy gradually increased with pregnancy and obviously exceeded the level of non-pregnancy after the middle and late pregnancy.

Pregnant women have physiological blood lipid elevation， and the reference range of normal values in this area is defined by percentile method， so as to provide clinicians with reasonable interpretation of blood lipid results at different stages of pregnancy.

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Meta-analysis of the efficacy and safety of Wuling capsule combined with Deanxit in the treatment of post-stroke depression

Min Jiao, Shuai Miao, Xiaoyan Xu

2021, 42(9):  685-690.  DOI: 10.3969/j.issn.2097-0005.2021.09.010

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To systematically evaluate the efficacy and safety of Wuling capsule combined with Deanxit in the treatment of post-stroke depression （PSD）， and to provide reference for the clinical application of drugs to prevent PSD.

Randomized controlled trial （RCTs） of Wuling caspule combined with Deanxit in the treatment of post-stroke depression were searched from CNKI，VIP，WanFang，PubMed and other datebases by electronic computer， and the deadline was September 2020.After the two researchers independently screened the literature， extracted the data and comprehensively evaluated the risk of bias included in this study， a Meta-analysis was performed on all the results using the Revnew 5.3 software.

A total of 23 RCTs were included， including 2059 patients （1033 patients in the experimental group and 1026 patients in the control group）.Meta-analysis results showed that the total effective rate of PSD patients in the experimental group was higher than that in the control group， with statistical significance （RR = 1.19，95%CI：1.14～1.24，P < 0.01）.The Hamilton depression scale （HAMD） score of patients with PSD reduction in the experimental group was significantly higher than that of the control group and was statistically significant （MD = －4.74，95%CI：－5.13～－3.75，P < 0.01）.The NIHSS score of patients with reduced PSD in the experimental group was higher than that in the control group and was statistically significant （MD = －2.33， 95% CI： －3.63～－1.02，P < 0.01）.The Barthel index （BI） score of patients with increased PSD in the experimental group was higher than that of the control group and was statistically significant （MD = 7.09， 95% CI： 4.78～9.41，P < 0.01）.The incidence of adverse reactions in the experimental group was significantly lower than that in the control group and was statistically significant （RR = 0.54，95% CI：0.41～0.70，P < 0.01）.

Compared with Deanxit， Wuling capsule combined with Deanxit can significantly improve the curative effect of PSD， improve the neurological function， daily life ability， and have fewer side effects.

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A meta-analysis of the relationship between vegetable intake and the risk of liver cancer

Xi Zhu, Xiu Wang

2021, 42(9):  691-696.  DOI: 10.3969/j.issn.2097-0005.2021.09.011

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To explore the relationship between vegetable intake and the risk of liver cancer， and to provide a scientific basis for disease prevention and treatment.

Case-control and cohort studies on vegetable intake and liver cancer risk published at home and abroad before November 2020 were systematically retrieved， and the effect value and 95%CI of liver cancer risk between the highest and lowest intake levels were combined to make analysis. Stata 11.0 and Revman 5.3 software were used for statistical analysis.

This study included 12 case-control studies and 9 cohort studies， involving a total of 1 961 683 subjects and 5023 liver cancer patients. Eating vegetables had a significant protective effect on liver cancer ［RR = 0.68， 95% CI （0.56， 0.82）， P < 0.01］. All strata in the subgroup analysis showed that the intake of vegetables could reduce the risk of liver cancer. The Begg′s test （P = 0.124） and the funnel plot indicated no publication bias.

The research results show that higher vegetable intake can reduce the risk of liver cancer. More prospective studies with larger sample sizes and good control of confounding factors are needed for further evaluation.

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The role of social capital in body size misperception among community-dwelling middle-aged and elderly people

Fangfang Hou, Yan Zhang, Jindong Wang, Dandan Miao, Guodong Shen

2021, 42(9):  697-701.  DOI: 10.3969/j.issn.2097-0005.2021.09.012

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To investigate the agreement between actual body size and the perceived body size， and the influencing factors of body size mis-perception among middle-aged and elderly residents in urban communities.

722 participants from two communities of Hefei City were surveyed with questionnaire. Height and weight were measured to assess their actual body size. Logistic regression was performed to identify whether social norms related to obesity and family attitude toward their body size were associated with body size mis-perception.

Self-reported prevalence of overweight and obesity were lower than the measured rates. The agreement between actual body size and the perceived body size was poor among male and female participants. The likelihood of mis-perception of their body size increased if the participants' family members held the attitude of "hoping me to gain weight" （OR：2.065，95%CI：1.250 ~ 3.411） and "don't care too much"（OR：1.775，95%CI：1.099 ~ 2.865）. On the contrary， higher meeting frequency with friends reduced the possibility of mis-perception （OR：0.85， 95%CI：0.752 ~ 0.962）.

Middle-aged and elderly residents in urban community tend to underestimate their body size. Within the components of bonding social capital， family's attitude can affect the correct judgement of the body size among middle-aged and elderly people. However， meeting with friends might be a protective factor. While enhancing health education for the weight management of middle-aged and elderly people， it is also necessary to provide suggestions through social capital perspectives.

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Analysis of the current state of health financing structure in China based on time and space

Suqing Wei, Ruowei Wang, Tingting Tian, Ying Shan, Tao Shi, Shixue Li

2021, 42(9):  702-705.  DOI: 10.3969/j.issn.2097-0005.2021.09.013

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To analyze the financing level and structure of the total health expenditure of 31 provincial administrative regions in Inland China from 2014 to 2018 from the perspective of time and space，and to discuss the regional differences.

The longitudinal changes of health financing level and structure in China were analyzed. Meanwhile， ArcGIS10.2 software was used to conduct spatial autocorrelation analysis on health financing structure data of 31 provinces.

From 2014 to 2018， China's health financing level was constantly improved， and the financing structure was optimized， but the regional differences between provinces were still obvious.

In the future， the focus of China's health work will still need to improve the level of health financing， optimize the financing structure， analyze specific roblems， pay attention to the regional differences of health financing structure， and solve the unfairness of health financing.

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Investigation， detection and analysis of an agnogenic foodborne disease outbreaks

Juanjuan Lu, Aiying Li, Jinhua Zheng, Hao Song

2021, 42(9):  706-708.  DOI: 10.3969/j.issn.2097-0005.2021.09.014

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To help handle emergency of foodborne disease by analyzing management of an agnogenic foodborne disease outbreaks and screening of pathogen.

We collected information of patients， made epidemiological investigation， did isolation， culture and molecular biological identification of pathogens， and analyzed.

After eating in canteen of Huangqian high school， 58 people showed symptoms of abdominal pain， diarrhea（86.21%，50/58）， nausea（53.45%，31/58）， vomiting（17.24%，10/58）. Some patients improved obviously with antibiotic therapy. Enterobacter cloacae was detected in 4 food samples， but others without pathogenic bacteria or conditioned pathogen， and test of Norovirus and COVID-19 was negative. Enterobacter cloacae liquid extracted from paired serums of 12 patients and food was all inagglutinable.

This emergency identified as a foodborne disease outbreaks of unknown reason due to comprehensive analysis of epidemiological features， clinical symptomsand laboratory testing. It is suggested that the food supervision department should strengthen thesupervision and management in campus，prevent similar incidents from happening again.

Review

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Study on adenosine and the pathogenesis of epilepsy

Mengqi Qi, Yuqin Lv, Wen Xu, Jingjun Zhang

2021, 42(9):  713-716.  DOI: 10.3969/j.issn.2097-0005.2021.09.017

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Epilepsy is one of the common diseases of the nervous system. In recent years， the synthesis， metabolic pathways and functions of adenosine in the brain， as well as the pathogenesis and treatment of epilepsy on adenosine， have been one of the hot research topics at home and abroad. In this paper， the research progress is reviewed.

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Moleular hydrogen， autophagy and oxidative stress

Keping Zhang, Longfei Gujing, Jianqiao Jiao, Xinran Liu, Fangshuo Yuan, Li Zhang, Jinguo Li, Zhaoqiang Zhang

2021, 42(9):  717-720.  DOI: 10.3969/j.issn.2097-0005.2021.09.018

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Molecular hydrogen is a highly reductive substance and has been widely studied as a selectively antioxidant. It can buffer the effects of reactive oxygen species （ROS） in tissue and has anti-oxidation， anti-inflammation， as well as anti-apoptotic functions. There are interaction effects between autophagy and ROS. Using molecular hydrogen can regulate the balance between oxidative stress and autophagy， regulates excessive autophagy and enhances insufficient autophagy， reduces levels of ROS， and maintains cellular homeostasis. In this brief review， it is aimed to elucidate related effects of molecular hydrogen for a reference for future studies.