Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata / 中国实验方剂学杂志

Objective:To detect the colony number of bacteria, yeasts and molds in fermentation process of Pinelliae Rhizoma Fermentata (PRF), microbial flora species, and quantitatively analyze the dynamic changes of four dominant microorganisms at different fermentation time points of PRF, so as to provide experimental basis for exploring the processing mechanism of PRF. Method:According to Pharmaceutical Standard Preparation of Traditional Chinese Medicine Prescription of Ministry of Health of the People's Republic of China (the 10th volume), PRF was processed. The samples at five different fermentation time points (0, 30, 60, 90, 120 h) of PRF were taken, the culturing, isolation and purification of bacteria, yeasts and molds were carried out with selective media, and the colonies were counted. Fluorescence quantitative polymerase chain reaction (PCR) technique was employed to conduct absolute quantification of Bacillus subtilis, Paecilomyces variotii, Byssochlamys spectabilis and Aspergillus niger. The recombinant plasmids of these 4 microorganisms were used as the standard substances, and the standard curves were prepared after dilution of multiple ratios, quantitative analysis was performed on these 4 microorganisms in five samples at different processing time points (0, 30, 60, 90, 120 h) of PRF. Result:During the fermentation process of PRF, the number of bacteria was low with smooth change, while molds and yeasts grew dramatically at the late stage of fermentation and reached 1×106 CFU·mL-1 at the end of fermentation. At 5 different fermentation time points, the copy numbers of Bacillus subtilis were 3.53×105, 7.56×104, 1.58×105, 1.90×106, 1.85×106 copies·g-1, the copy numbers of Paecilomyces variotii were 0, 0, 0, 3.45×107, 4.15×108 copies·g-1, the copy numbers of Byssochlamys spectabilis were 0, 0, 0, 1.04×108, 2.28×108 copies·g-1, the copy numbers of Aspergillus niger were 0, 0, 9.48×105, 1.47×106, 7.56×106 copies·g-1, respectively. Conclusion:The change trend of microflora in the fermentation process of PRF can be reflected by the dynamic change of four dominant microorganisms, and molds may play an important role in the processing of PRF. Fluorescence quantitative PCR technique has the advantages of rapidity, sensitivity, good repeatability and high specificity, it is suitable for exploring processing mechanism of PRF.

Effect of protein intervention on amino acid metabolism spectrum of Qi and Yin deficiency type 2 diabetic rats / 中国中药杂志

To study the effect of plant protein and animal protein on amino acid metabolism spectrum of Qi and Yin deficiency type 2 diabetic rats. 110 male SD rats were randomly divided into blank group (n=10), diabetic model group (n=20), disease-symptoms group (n=80). The rats of blank group received ordinary feeding, while other groups were fed with high sugar and fat diets. During the whole process of feeding, rats of disease-symptoms group were given with Qingpi-Fuzi (15.75 g•kg⁻¹) once a day through oral administration. Five weeks later, the rats were given with a low dose of STZ (40 mg•kg⁻¹) by intraperitoneal injection to establish experimental diabetic models. Then the models were randomly divided into disease-symptoms group 1 (Qi and Yin deficiency diabetic group, 15.75 g•kg⁻¹), disease-symptoms group 2 (plant protein group, 0.5 g•kg⁻¹), disease-symptoms group 3 (animal protein group, 0.5 g•kg⁻¹), disease-symptoms group 4 (berberine group, 0.1 g•kg⁻¹). The drugs were given for 4 weeks by gavage administration. After 4 weeks of protein intervention, the abdominal aortic blood was collected and serum was isolated to analyze its free amino acid by using AQC pre-column derivatization HPLC and fluorescence detector. Four weeks after the protein intervention, plant protein, animal protein and berberine had no obvious effect on body weight and blood sugar in type 2 diabetic rats. As compared with animal protein group, histidine and proline（P<0.01）, serine, glycine, threonine, alanine, tyrosine, valine, methionine, bright+isoleucine, phenylalanine and lysine（P<0.05）changed a lot in rats serum of plant protein group.The results showed that gavage administration of protein would produce effects on amino acid metabolism of Qi and Yin deficiency type 2 diabetic SD rats. Symbolic differential compounds could be found through metabonomics technology, providing experimental basis for early warning of type 2 diabetes and diagnosis of Qi and Yin deficiency syndrome.

Identification of dominant microbes from traditional Chinese medicine Pinelliae Rhizoma Fermentata / 中国中药杂志

To investigate the microbial species, amount changes as well as the isolation and identification of domain strains at different fermentation time points of Pinelliae Rhizoma Fermentata, and provide basis for exploring the mechanism of Pinelliae Rhizoma Fermentata processing. Five samples were chosen at the time points (0, 18, 36, 54, 72 h) of Pinelliae Rhizoma Fermentata processing. Bacteria, mold and yeast from the samples were cultured; their colonies were counted, and the dominant strains were isolated and purified. The dominant bacteria and dominant fungi were identified by 16S rDNA and 26S rDNA sequencing respectively. The results showed that the bacteria count was low with slow and smooth changes in the fermentation process;while mold and yeast grew dramatically after 54 h culturing and reached 1×107 CFU•mL⁻¹ at the end of fermentation. Through the NCBI homology alignment and phylogenetic tree construction, the dominant bacteria were identified as Streptomyces sp., Bacillus pumilus, B. subtilis, B. aryabhattai and other Bacillus sp.; the dominant yeast was identified as Meyerozyma guilliermondii; the dominant mold were identified as Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger in the processing of Pinelliae Rhizoma Fermentata. The results indicated that multiple microbe species, especially yeast and mold, played a role in the fermentation processing of Pinelliae Rhizoma Fermentata. M. guilliermondii, P. variotii, P. variotii and A. niger and Bacillus sp. can be the crucial factors in the processing of Pinelliae Rhizoma Fermentata.

Effects of caffeine citrate on myelin basic protein in neonatal rats with hypoxic-ischemic brain damage / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effects of caffeine citrate on myelin basic protein (MBP) expression in the cerebral white matter of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the related mechanism.</p><p><b>METHODS</b>Forty-eight seven-day-old Sprague-Dawley neonatal rats were randomly assigned to 3 groups: sham operation (n=16), HIBD (n=16) and HIBD+caffeine citrate (n=16). The rats in the HIBD and HIBD+caffeine citrate groups were subjected to left common carotid artery ligation, and then were exposed to 80 mL/L oxygen and 920 mL/L nitrogen for 2 hours to induce HIBD. The rats in the sham operation group were only subjected to a sham operation, without the left common carotid artery ligation or hypoxia exposure. Caffeine citrate (20 mg/kg) was injected intraperitoneally before hypoxia ischemia (HI) and immediately, 24 hours, 48 hours and 72 hours after HI. The other two groups were injected intraperitoneally with an equal volume of normal saline at the corresponding time points. On postnatal day 12, the expression of MBP in the left subcortical white matter was detected by immunohistochemistry, and the levels of adenosine A1 receptor mRNA and A2a receptor mRNA in the left brain were detected by real-time PCR.</p><p><b>RESULTS</b>The expression of MBP in the left subcortical white matter in the HIBD group was lower than in the sham operation group (P<0.05). The MBP expression in the HIBD+caffeine citrate group was significantly higher than in the HIBD group, but was still lower than the sham operation group (P<0.05). Real-time PCR showed that the adenosine A1 receptor mRNA expression was significantly higher in the HIBD group than in the sham operation group, and it was significantly lower in the HIBD+caffeine citrate group than in the HIBD group (P<0.05).</p><p><b>CONCLUSIONS</b>Caffeine citrate can improve brain white matter damage following HIBD in neonatal rats and the protection mechanism might be related with the down-regulation of adenosine A1 receptor expression.</p>

Effects of erythropoietin on neuronal proliferation and apoptosis in neonatal rats after infection-induced brain injury / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effects of erythropoietin (EPO) on the neuronal proliferation and apoptosis in neonatal rats after infection-induced brain injury and the neuroprotective mechanism of EPO in neonatal rats with infection-induced brain injury.</p><p><b>METHODS</b>Twenty-six two-day-old neonatal rats were randomly divided into 3 groups: control group (intraperitoneally given an equal volume of normal saline), lipopolysaccharide (LPS) group (intraperitoneally given LPS 0.6 mg/kg), and EPO group (intraperitoneally given LPS 0.6 mg/kg and EPO 5 000 U/kg). These groups were injected with respective drugs for 5 consecutive days. Meanwhile, each group was intraperitoneally injected with 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg) once a day for 5 consecutive days. The expression of BrdU and cleaved Caspase-3 in the hippocampal dentate gyrus was detected by immunohistochemistry at 24 hours after the last injection.</p><p><b>RESULTS</b>The number of neuronal cells in the hippocampal dentate gyrus in the LPS and EPO groups was significantly greater than in the control group (P<0.05), but there was no significant difference between the LPS and EPO groups. The EPO group had a significantly higher number of BrdU-positive cells in the subgranular zone of hippocampal dentate gyrus than the LPS group (51±9 vs 29±6; P<0.05), but a significantly lower number of BrdU-positive cells than the control group (51±9 vs 67±12; P<0.05). The EPO group had a significantly lower number of cleaved Caspase-3-positive cells in the subgranular zone of hippocampal dentate gyrus than the LPS group (27.9±1.5 vs 34.0±1.3; P<0.05), but a significantly higher number of cleaved Caspase-3-positive cells than the control group (27.9±1.5 vs 21.0±1.7; P<0.05).</p><p><b>CONCLUSIONS</b>EPO can promote hippocampal neuronal proliferation and reduce neuronal apoptosis in neonatal rats after infection-induced brain injury.</p>

Risk factors of bronchopulmonary dysplasia in preterm infants with different severities / 中华实用儿科临床杂志

Objective To improve the prevention and treatment of bronchopulmonary dysplasia(BPD) in preterm infants,and the clinical risk factors of premature neonates with different severities of BPD were investigated.Methods A total of 139 cases among preterm infants who were admitted to NICU in the Third Affiliated Hospital of Zhengzhou University from Jan.2007 to Dec.2011 were analyzed retrospectively.The history of birth and mother pregnancy,clinical treatment,prognosis and complication of mild,moderate and severe BPD according to clinical diagnostic criteria were analyzed,respectively.Results Of the total 139 premature neonates,61 cases were diagnosed as mild BPD,48 cases as moderate BPD and 30 cases as severe BPD.No significant differences were found in gender,birth times,fertilization,delivery mode,the percentage of fetal distress and neonatal resuscitation,maternal age,the percentage of pregnancy-induced hypertension,the percentage of antenatal corticosteroids administration and postnatal pulmonary surfactant and combined with patent ducts arteries among the different groups(all P ＞ 0.05).With the increasing severity of BPD,the birth weight and gestational age were decreasing,the percentage of the infants with Apgar 1 minute score ≤7,premature rupture of membranes ≥ 8 hours,maternal perinatal infection,meconium-stained amniotic fluid were increasing(all P ＜ 0.05).And mechanical ventilation,the time of using oxygen,and the percentage of trachea cannula intubation ≥2 times,indwelling gastric tube and red blood cells transfusing,the positive rate of sputum cultures and the blood culture were also increased with the increasing severity of BPD(all P ＜ 0.05).Conclusions Preventing of preterm delivery,control and reduce antenatal and postnatal infection,shorten the duration of mechanical ventilation and usage of oxygen are key factors to reduce BPD and severities in neonatal infants.

Effects of bacterial lipopolysaccharide and normobaric hyperoxia on immature brain development of neonatal rat / 中华实用儿科临床杂志

Objective To investigate the effects of lipopolysaccharide (LPS) and / or normobaric hyperoxia on brain development of neonatal rat and the possible mechanisms.Methods One hundred and twenty postnatal day 2 (P2) SD rats were randomly assigned into 4 groups:air group,LPS group,hyperoxia group,LPS + hyperoxia group.General condition and body weight of the rats in each group were observed and recorded every day.The expression of active Caspase-3 and nuclear factor-κappaB P65 (NF-κB P65) in the brain were detected by immunohistochemistry staining on P7,and the level of IL-6 and 8-iso-PGF2α in the brain homogenate were measured by enzyme-linked immunosorbent assay(ELISA).The expression of myelin basic protein (MBP) in the brain was detected by immunohistochemistry staining on P12.Results The expressions of Caspase-3 and NF-κB P65 had the same trends:the number of positive cells from high to low was in LPS + hyperoxia group,LPS group/hyperoxia group,air group.There were significant differences between the first three groups and air group(all P ＜ 0.05).There were also significant differences between LPS + hyperoxia group and LPS group or hyperoxia group(all P ＜0.01).MBP in the brain had the completely reverse expression:from high to low order was in air group,hyperoxia group,LPS group,LPS + hyperoxia group.There were significant differences between the last three groups and air group (all P ＜ 0.05).There were also significant differences between LPS + hyperoxia group and LPS group or hyperoxia group(all P ＜0.01).The level of IL-6 in the brain from high to low order respectively was in LPS + hyperoxia group,LPS group,hyperoxia group,air group;and 8-iso-PGF2α was also in LPS + hyperoxia group,hyperoxia group,LPS group,air group,Significant differences were found among the four groups (all P ＜ 0.05).Conclusions Both postnatal infection and normobaric hyperoxia may induce premature rat brain injury,and increase the number of apoptosis cell and reduce the expression of MBP.The combination of infection and normobaric hyperoxia may aggravate the degree of brain damage of neonatal rat.NF-κB pathway mediated by Toll-like receptor may be involved in inflammation and oxidative stress,and may mediate Caspase-3 related apoptosis of nerve cell and white matter injury.

Risk factors for periventricular-intraventricular hemorrhage in premature infants treated with mechanical ventilation / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study risk factors for periventricular-intraventricular hemorrhage (PVH-IVH) in premature infants treated with mechanical ventilation.</p><p><b>METHODS</b>A total of 205 premature infants who were admitted to the neonatal intensive care unit (NICU) and treated with mechanical ventilation between January 2009 and December 2011 were enrolled. They were classified into PVH-IVH and non-PVH-IVH groups according to the results of head ultrasonography performed at 3 to 7 days after birth. Single factor and multivariate logistic regression analysis were used to identify risk factors for PVH-IVH.</p><p><b>RESULTS</b>Single factor analysis indicated 9 factors associated with the development of PVH-IVH, including a gestational age of <32 weeks, a birth weight of <1500 g, intrauterine distress, severe asphyxia, vaginal delivery, maternal perinatal infection, premature rupture of membranes (PROM) at ≥8 hours, mechanical ventilation duration of ≥7 days and ventilator-associated pneumonia (VAP) (P<0.05). Multivariate logistic regression analysis showed that a birth weight of <1500 g (OR=2.665), intrauterine distress (OR=2.177), severe asphyxia (OR=5.653), maternal perinatal infection (OR=4.365) and VAP (OR=2.299) were independent risk factors for the development of PVH-IVH (P<0.05).</p><p><b>CONCLUSIONS</b>Very low birth weight, intrauterine distress, severe asphyxia, maternal perinatal infection and VAP are closely associated with an increased risk of PVH-IVH in premature infants treated with mechanical ventilation. These clinical risk factors should be given more attention in the prevention of PVH-IVH.</p>