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ObjectiveTo construct traditional Chinese medicine （TCM） diagnostic scale of turbid toxin syndrome in order to provide corresponding reference for the standardization of TCM syndromes and studies. MethodsWe systematically searched the Chinese Medical Dictionary （CMD）， China Knowledge Network （CNKI）， Wanfang Data Knowledge Service Platform （WF） and VIP database for TCM classics and modern literature on turbid toxin syndrome， and initially screened the four diagnosis information of turbid toxin syndrome， established a pool of information entries， and conducted a cross-sectional clinical survey. Discrete trend method， correlation coefficient method， Cronbach's coefficient method， and factor analysis method were applied to objectively screen the entries. The diagnostic scale of turbid toxin syndrome were constructed through three rounds of Delphi method expert survey to determine the scale entries， using hierarchical analysis to get the judgement matrix scores and relative weight of each entry， after passing consistency test and then isometric expansion of the relative weight of the entries to get the weight of each entry and assign the value. ResultsA total of 35 articles were included， 45 entries were obtained after the initial screening. After the clinical investigation， 12 entries were not suitable by the discrete trend method， 23 entries not suitable by correlation coefficient method， 13 entries by the internal consistency screening were removed with the Cronbach's alpha coefficient rising， and 10 entries not suitable by the factor analysis method. Twenty-two entries were retained after objective screening by the combined use of the four statistical methods. The positive coefficients of experts in the three rounds of Delphi method of expert consultation were 96.67%， the coefficients of expert authority were 0.834， 0.856， and 0.867， and the coefficients of co-ordination were 0.126， 0.326， and 0.312， respectively. After consulting with clinical experts， and three rounds of Delphi method survey and hierarchical analysis method weight assignment， the diagnostic scale entries of turbid toxin syndrome were finally established. Primary symptoms： dark red or purple and dusky tongue， yellowish greasy or dry coating （10 points）； sticky and unpleasant stools （8 points）； disharmony of tastes including halitosis， sticky and greasy taste in the mouth， dry mouth and bitter taste in the mouth （6 points）； unfavourable or yellowish or red urination （5 points）； and dark complexion （4 points）. Secondary symptoms： heavy body （3 points）； dizziness （3 points）； profuse， sticky， foul-smelling secretions （2 points）； wiry and slippery， or slippery， or slippery and rapid pulse （2 points）； feeling of hardness in the abdomen （1 point）. ConclusionUsing Delphi method combined with the hierarchical analysis method， combining qualitative and quantitative study， a diagnostic scale of turbid toxin syndrome was initially developed.
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Objective:To construct and identify the lentiviral vector of adenosine RNAi-adenosine A2a receptor (A2aR) in rats.Methods:Three pairs of short hairpin RNA(shRNA)-A2aR sequences (shRNA-A2aR 1, shRNA-A2aR 2, shRNA-A2aR 3) were designed, and three pairs of double-stranded shRNA oligos were respectively inserted into the shRNA virus vector to gain three kinds of shRNA lentiviral recombinant plasmid.The recombinant plasmid, packaging vector, and shuttle vector were co-transfected into 293T cells to obtain virus liquid.The experiment was performed in two parts.Part Ⅰ The rat primary cardiomyocytes were divided into 3 groups ( n= 6 each) by a random number table method: vehicle group (V group), shRNA-A2aR 1 group and shRNA-A2aR 3 group.Each group was transfected with virus solution of MOI 10 for 48 h. The expression of A2aR was detected by Western blot to select the most efficient lentivirus vector.Part Ⅱ The cardiomyocytes were randomly divided into 6 groups ( n=36 each): vehicle group (V group), MOI5 group, MOI10 group, MOI15 group and MOI20 group.Each group was transfected with the corresponding MOI virus liquid (the most effective lentivirus vector). At 24, 48, and 72 h of transfection, the cell viability and cell death were observed with a fluorescent microscope, and the A2aR expression was detected by Western blot to determine the interference efficiency. Results:Part Ⅰ Two types of shRNA-A2aR lentiviral vectors (shRNA-A2aR 1, 3) were successfully constructed, among which shRNA-A2aR 3 virus solution with a titer of 3.5×10 8 TU/ml had the best effect.Compared with group V and group shRNA-A2aR 1, the expression of A2aR in cardiomyocytes was significantly down-regulated ( P<0.01), and the interference efficiency of shRNA-A2aR 3 was 73% in shRNA-A2aR 3 group.Part Ⅱ shRNA-A2aR 3 was selected to screen out the transfection plan.The cell survival rate in each group was more than 85% at 24 h of transfection, the cell survival rate was more than 80% at 48 h of transfection in MOI5 and MOI10 groups; the cell survival rate in each group was less than 70% at 72 h of transfection.Under an inverted fluorescent microscope, a slightly lower fluorescence density was found in MOI5 group, the fluorescent density was higher and the cell condition was better at 48 h of transfection in MOI10 group and at 24 h of transfection in MOI20 group, and the cardiomyocyte viability was significantly decreased, and dead cells were increased at 72 h of transfection in each group.The results of Western blot showed that the interference efficiency at 48 h of transfection in MOI10 group, 48 h in MOI15 group, 24 and 48 h in MOI20 group was all > 70%. Conclusion:MOI of 10, transfection for 48 h or MOI of 20, transfection for 24 h is the optimal transfection protocol.
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Objective:To evaluate the effect of valproic acid on the expression of M1/M2 microglia in the prefrontal cortex of rats with neuropathic pain (NP).Methods:Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 6-7 weeks, weighing 200-230 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (group S), group NP, and valproic acid group (group V). The NP model was established by ligation of the L 5 spinal nerve (SNL) of anesthetized rats.Valproic acid 300 mg/kg was intraperitoneally injected immediately after SNL and every day after ligation, once a day, for 3 consecutive days in group V, while the equal volume of normal saline was given instead of valproic acid in S and NP groups.The mechanical paw withdrawal threshold (MWT) was measured before ligation and at 1, 3, 7, 14, 21 and 28 days after ligation.Sucrose preference test and forced-swim test were performed on day 28 after ligation.After the end of the behavior test, the prefrontal cortex was removed for determination of the expression of cluster of differentiation (CD) 16 and CD206 by Western blot.The ratio of CD206/CD16 was calculated. Results:Compared with group S, the MWT at each time point after ligation and rate of preference for sucrose were significantly decreased, the duration of immobility in forced-swim test was prolonged, the expression of CD16 and CD206 was up-regulated, and the ratio of CD206/CD16 was decreased in group NP ( P<0.05). Compared with group NP, the MWT at each time point after ligation and rate of preference for sucrose were significantly increased, the duration of immobility in forced-swim test was shortened, the expression of CD16 was down-regulated, the expression of CD206 was up-regulated, and the ratio of CD206/CD16 was increased in group V ( P<0.05). Conclusion:The mechanism by which valproic acid improves depression may be related to promoting the expression of M2 microglia and inhibiting the expression of M1 microglia in the prefrontal cortex of rats with NP.
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Objective:To evaluate the role of histone deacetylase 6 (HDAC6) in the maintanence of neuropathic pain (NP) and the relationship with myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) signaling pathway in the rats.Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-260 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), sham operation group (group S), NP group and NP plus HDAC6 inhibitor ACY-1215 group (group NP+ ACY). The rat model of NP was established by ligating the L 5 spinal nerve in anesthetized rats.The L 5 spinal nerve was only exposed without ligation in group S. In NP+ ACY group, ACY-1215 25 mg/kg was intraperitoneally injected daily for 21 days after the end of model establishing.The equal volume of solvent was intraperitoneally injected in S and NP groups, and group C was reared normally.The mechanical paw withdrawal threshold (MWT) was measured on 3 days before establishing the model (T 0), the day before establishing the model (T 1) and 1, 3, 7, 10, 14 and 21 days after establishing the model (T 2-7). The rats were sacrificed after measurement of MWT on day 21 after ligation, and the spinal dorsal horn tissues of L 4-6 were removed for determination of the expression of MyD88, NF-κB and phosphorylated NF-κB (p-NF-κB) (by Western blot) and expression of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) mRNA (by real-time polymerase chain reaction). Results:Compared with C and S groups, the MWT was significantly decreased at T 2-7, and the expression of MyD88, NF-κB, p-NF-κB, TNF-α mRNA and IL-1β mRNA was up-regulated in NP and NP+ ACY groups ( P<0.05). Compared with group NP, the MWT was significantly increased at T 5-7, and the expression of MyD88, NF-κB, p-NF-κB, TNF-α mRNA and IL-1β mRNA was down-regulated in group SNL+ ACY ( P<0.05). Conclusion:HDAC6 activation is involved in the maintanence of NP, which is related to activating MyD88/NF-κB signaling pathway in the rats.
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Objective@#To explore the status of sugar sweetened beverages(SSBs) and its associated factors among primary and middle school students in Shenzhen, providing reference for nutrition and health education.@*Methods@#A random number table and convenience sampling method was used to select 40 135 primary and middle school students aged 6-18 years in Shenzhen. Data was collected to investigate their SSBs knowledge and associated factors.@*Results@#The proportion of SSBs knowledge score less than 60 points, between 60-79 points and 80-100 points were 5.6%, 41.9% and 52.4%. Multivariate analysis showed that age(4-6 grade, junior middle and high school), gender(female), parents education(high school or vocational schools, colleges and universities as bachelors, masters or doctors), students pay attention to the ingredient list (seldom, none), parental dissuasion or reward behavior (only forced dissuasion, giving no dissuasion, occasional reward, no reward), and the storing beverages at home (seldom, none) were associated with total SSBs knowledge score and milk containing beverage knowledge score ( β =-0.79，-1.19，-1.74，0.58， 1.20 ，1.81，2.98，3.13，2.70，4.85，6.34,6.41，-0.99，-0.78，-1.81，-2.40，5.85，6.26，0.61，1.92， P <0.05). Age(4-6 grade, junior middle and high school), gender(female), father s education background(with colleges and universities as bachelor, masters or doctors), mother s education background(high schools or vocational schools, colleges and universities as bachelors, masters or doctors), parents dissuation behaviors(giving no dissuation), parents rewarding behavior(seldom, none), storing beverages at home(seldom, none) were associated with the total SSBs knowledge score and milk containing beverage knowledge score( β =-0.68， -0.92 ，-0.49，0.26，0.51，1.05，1.09，0.90，1.93，2.62，2.55，-0.68，0.93，1.13，0.21，0.92， P <0.05).@*Conclusion@#Primary and middle school students have moderate to high level of SSBs knowledge. It is necessary for students and their parents to learn more SSBs related nutrition healthy knowledge, and to reduce home availability of SSBs.
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Objective@#To explore the status of sugar sweetened beverages(SSBs) and its associated factors among primary and middle school students in Shenzhen, providing reference for nutrition and health education.@*Methods@#A random number table and convenience sampling method was used to select 40 135 primary and middle school students aged 6-18 years in Shenzhen. Data was collected to investigate their SSBs knowledge and associated factors.@*Results@#The proportion of SSBs knowledge score less than 60 points, between 60-79 points and 80-100 points were 5.6%, 41.9% and 52.4%. Multivariate analysis showed that age(4-6 grade, junior middle and high school), gender(female), parents education(high school or vocational schools, colleges and universities as bachelors, masters or doctors), students pay attention to the ingredient list (seldom, none), parental dissuasion or reward behavior (only forced dissuasion, giving no dissuasion, occasional reward, no reward), and the storing beverages at home (seldom, none) were associated with total SSBs knowledge score and milk containing beverage knowledge score ( β =-0.79，-1.19，-1.74，0.58， 1.20 ，1.81，2.98，3.13，2.70，4.85，6.34,6.41，-0.99，-0.78，-1.81，-2.40，5.85，6.26，0.61，1.92， P <0.05). Age(4-6 grade, junior middle and high school), gender(female), father s education background(with colleges and universities as bachelor, masters or doctors), mother s education background(high schools or vocational schools, colleges and universities as bachelors, masters or doctors), parents dissuation behaviors(giving no dissuation), parents rewarding behavior(seldom, none), storing beverages at home(seldom, none) were associated with the total SSBs knowledge score and milk containing beverage knowledge score( β =-0.68， -0.92 ，-0.49，0.26，0.51，1.05，1.09，0.90，1.93，2.62，2.55，-0.68，0.93，1.13，0.21，0.92， P <0.05).@*Conclusion@#Primary and middle school students have moderate to high level of SSBs knowledge. It is necessary for students and their parents to learn more SSBs related nutrition healthy knowledge, and to reduce home availability of SSBs.