ObjectiveTo investigate the mechanism of Hei Xiaoyaosan （HXS） in ameliorating cognitive impairment in the rat model of Alzheimer's disease （AD） by regulating the adenosine 5'-monophosphate-activated protein kinase （AMPK）/sterol regulatory element-binding protein 2 （SREBP2）/proprotein convertase subtilisin/kexin type 9 （PCSK9）/low-density lipoprotein receptor-related protein 1 （LRP-1） signaling pathway to clear amyloid-β （Aβ） from the peripheral circulation. MethodsNinety 8-week-old specific pathogen-free （SPF）-grade male Sprague-Dawley （SD） rats were acclimatized for 1 week. After acclimatization， 10 rats were randomly selected as the blank control group， and the remaining 80 rats were prepared for surgical modeling. Among the 80 rats for modeling， 70 were anesthetized and injected with 1 μL of amyloid-β_1-42 （Aβ_1-42） solution into the bilateral hippocampal CA1 region for the modeling of AD， and the remaining 10 were assigned to the sham operation group， which received an injection of 1 μL normal saline at the same anatomical site. The successfully modeled rats were randomly assigned into 6 groups （n=10 per group）： a model group， a donepezil hydrochloride group， an acadesine （AICAR， an AMPK agonist） group， and high-， medium-， and low-dose HXS groups. After 42 consecutive days of administration intervention， the Morris water maze test and T-maze test were performed to evaluate the learning and memory abilities of rats. Fluoro-Jade B （FJB） staining was conducted to observe pathological changes of neurons in the rat hippocampus， and thioflavin S staining to quantify the area of Aβ deposition in the hippocampal region. Immunofluorescence double staining （IF-DS） was performed to detect the expression of LRP-1 and Aβ signals in the liver tissue. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of Aβ_1-42 and soluble LRP-1 （sLRP-1） in the serum. Western blot was performed to determine the protein levels of AMPK， phosphorylated AMPK （p-AMPK）， SREBP2， PCSK9， and LRP-1 in the liver tissue. Real time polymerase chain reaction（Real-time PCR） was adopted to assess the mRNA levels of AMPK， SREBP2， PCSK9， and LRP-1 in the liver tissue. ResultsCompared with the blank control group， the model group showed increased total swimming distance on day 6 and path length in the target quadrant on day 7 （P<0.01）， a decreased correct alternation rate in the T-maze test （P<0.01）， disarrangement of hippocampal neurons， increased FJB-positive cells （P<0.01）， enlarged area of Aβ deposition （P<0.01）. reduced LRP-1 fluorescence signals and enhanced Aβ fluorescence signals in the liver tissue （P<0.01）， elevated serum level of Aβ_1-42， declined serum level of sLRP-1 （P<0.01）， downregulated protein levels of p-AMPK and LRP-1 as well as mRNA levels of AMPK and LRP-1， and upregulated protein and mRNA levels of SREBP2 and PCSK9 in the liver tissue （P<0.01）. Compared with the model group， the high-dose HXS group， donepezil hydrochloride group， and AICAR group exhibited reduced total swimming distance and target quadrant path length （P<0.05， P<0.01）， an increased correct alternation rate in the T-maze test （P<0.05）， decreased FJB-positive cells （P<0.01）， reduced Aβ deposition （P<0.01） in the hippocampal tissue， enhanced LRP-1 signals and weakened Aβ signals in the liver tissue （P<0.01）， a declined serum level of Aβ_1-42 and an elevated serum level of sLRP-1 （P<0.01）， upregulated protein levels of p-AMPK and LRP-1 as well as mRNA levels of AMPK and LRP-1， and downregulated protein and mRNA levels of SREBP2 and PCSK9 in the liver tissue （P<0.01）. ConclusionHei Xiaoyaosan can regulate the AMPK/SREBP2/PCSK9/LRP-1 signaling pathway to enhance the clearance of peripheral Aβ by the liver tissue and reduce Aβ deposition in the brain， thereby improving the learning and memory abilities of AD rats.

ObjectiveTo investigate the role and mechanism of Hei Xiaoyaosan in regulating asparaginyl endopeptidase （AEP） to inhibit tau protein （tau） phosphorylation， amyloid-beta protein （Aβ） deposition， and neuroinflammation in the prevention and treatment of Alzheimer's disease （AD）. MethodsNinety SPF-grade SD male rats of 8 weeks old were selected and randomly grouped as follows： 10 rats in the blank group， 10 rats in the sham group （bilateral hippocampus injected with 1 μL normal saline）， and 70 rats in the modeling group （bilateral hippocampus injected with 1 μL Aβ_1-42 solution for the modeling of AD）. Fifty successfully modeled rats were selected and randomly assigned into the model， donepezil hydrochloride （positive control， 0.45 mg·kg^-1）， and high-， medium-， and low-dose （15.30， 7.65， and 3.82 g·kg^-1， respectively） Hei Xiaoyaosan groups. Rats were administrated with corresponding agents by gavage once a day for 42 days. Morris water maze and Y maze tests were carried out to assess the cognitive abilities of rats. Hematoxylin-eosin （HE） staining was employed to observe morphological changes in hippocampal neurons， and immunohistochemistry （IHC） was employed to assess phosphorylation （p）-tau and Aβ expression in the hippocampal tissue of rats. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of interleukin-1 beta （IL-1β）， interleukin-18 （IL-18）， and Aβ_1-42 in the hippocampal tissue. Western blot was employed to determine the protein levels of AEP， tau， phosphorylated tau （p-tau）， α-Syn （α-Syn）， phosphorylated α-Syn （p-α-Syn）， Toll-like receptor 2 （TLR2）， and NOD-like receptor thermal protein domain associated protein 3 （NLRP3）. Real time polymerase chain reaction （Real-time PCR） was employed to determine the mRNA levels of AEP， tau， α-Syn， TLR2， and NLRP3. ResultsCompared with the blank group， the model group showed prolonged escape latency， a decreased percentage of time spent in the platform quadrant， and a decreased alternation rate （P<0.01）， a reduced number， shrinkage， and increased vacuoles of hippocampal neurons， increased expression of p-tau and Aβ in the hippocampus （P<0.01）， elevated levels of IL-1β， IL-18， and Aβ_1-42， up-regulated protein and mRNA levels of AEP， tau， α-Syn， TLR2， and NLRP3， and up-regulated protein levels of p-tau and p-α-Syn （P<0.01）. Compared with the model group， the positive control group and the high-dose Hei Xiaoyaosan group exhibited shortened escape latency （P<0.01）， an increased percentage of retention time in the target quadrant， an increased alternation response rate （P<0.05， P<0.01）， alleviated neuronal shrinkage， regular morphology and arrangement of neurons in the hippocampal tissue， lowered levels of p-tau and Aβ in the hippocampal tissue （P<0.05， P<0.01）， declined levels of IL-1β， IL-18， and Aβ_1-42， downregulated mRNA and protein levels of AEP， tau， α-Syn， TLR2， and NLRP3， and downregulated protein levels of p-tau and p-α-Syn （P<0.05， P<0.01）. ConclusionHei Xiaoyaosan can protect hippocampal neurons and ameliorate cognitive impairment in the rat model of AD. Specifically， it may inhibit AEP to regulate tau phosphorylation， Aβ deposition， and neuroinflammation.

ObjectiveTo investigate the effect and mechanism of Hei Xiaoyaosan in preventing and treating Alzheimer's disease （AD） by activating autophagy through regulating the adenosine monophosphate-activated protein kinase （AMPK）/UNC-51 like autophagy activating kinase 1 （ULK1） signaling pathway. MethodsNinety 8-week-old male SPF-grade SD rats were selected and randomized into two groups： a blank control group （n=10） and a modeling group （n=80）. From the modeling group， 10 rats were randomly assigned as a sham group （bilateral hippocampal injection of 1 μL normal saline）， while the remaining 70 rats were injected with 1 μL Aβ_1-42 solution bilaterally into the hippocampus to replicate the AD model. The Morris water maze test was conducted to screen the successfully modeled rats， which were then randomly allocated to the model， donepezil hydrochloride， 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside （AICAR， an AMPK agonist）， and high-， medium-， and low-dose Hei Xiaoyaosan groups， with 10 rats per group. After 42 days of drug intervention， the Morris water maze and open field tests were conducted to assess learning and memory abilities and motor function. Pathological changes in the hippocampal tissue were observed by Nissl staining， and autophagy-related pathological damage was examined via transmission electron microscopy （TEM）. The expression of LC3 in the hippocampus was observed by immunofluorescence staining. The protein levels of AMPK， phosphorylate （p）-AMPK， ULK1， p-ULK1， p62， and autophagy related 7 （Atg7） were determined by Western blot， while the mRNA levels of AMPK and ULK1 were measured by Real-time polymerase chain reaction （Real-time PCR）. ResultsCompared with the blank control group， the model group showed increased escape latency （P<0.01） and decreased platform crossings （P<0.01） on day 6 in the water maze test， reduced total distance traveled and average speed in the open field test （P<0.01）， disarranged neurons with Nissl body loss and evident autophagic damage， weakened LC3 fluorescence signal （P<0.01）， downregulated protein levels of p-AMPK， p-ULK1， and Atg7 and mRNA levels of AMPK and ULK1 in the hippocampus （P<0.01）， and upregulated protein level of p62 （P<0.01）. Compared with the model group， the positive drug group， high-dose Hei Xiaoyaosan group， and AICAR group showed decreased escape latency （P<0.05， P<0.01） and increased platform crossings （P<0.01） on day 6， and increased total distance traveled and average speed in the open field test （P<0.05）. The positive drug group， high- and medium-dose Hei Xiaoyaosan groups， and the AICAR group showed alleviated cellular structural damage， regular neuronal arrangement， increased Nissl bodies， and orderly distribution of autophagosomes. Furthermore， the LC3 fluorescence intensity was enhanced in the positive drug group， high- and medium-dose Hei Xiaoyaosan groups， and the AICAR group （P<0.01）. The positive drug group， high-dose Hei Xiaoyaosan group， and the AICAR group exhibited upregulated protein and mRNA levels of p-AMPK and p-ULK1 （P<0.05， P<0.01）， upregulated protein level of Atg7 （P<0.05， P<0.01）， and downregulated protein level of p62 （P<0.01）. ConclusionHei Xiaoyaosan can improve learning and memory abilities and alleviate pathological status in the rat model of AD by regulating the AMPK/ULK1 signaling pathway， enhancing cellular autophagy， and repairing damaged nerve cells.

OBJECTIVE To study the toxicokinetics and tissue distribution characteristics of alpha-amanitin in rats.METHODS The tail venous blood was collected from SD rats before and 5,10,20,30 and 45 min,1,1.5,2.5,4 and 8 h after intraperitoneal injection of alpha-amanitin(1.5 mg·kg-1),and the concentration of alpha-amanitin in blood was determined by liquid chromatography-mass spectrometry(LC-MS/MS).DAS 2.0 software was used to analyze and plot the drug-time curve with toxicokinetic parame-ters.Based on the toxicokinetics results,18 SD rats were randomly divided into three groups.The rats were sacrificed,and left ventricular arterial(LVA)blood and 9 types of tissue samples involving the heart,liver,spleen,lung,kidney,whole brain,small intestine,stomach wall and testis were collected 15 min,40 min and 2.5 h after dosing,and the concentrations of alpha-amanitin were measured by LC-MS/MS to obtain the tissue distribution results of alpha-amanitin in SD rats.RESULTS Toxicokinetics studies revealed that the peak blood concentration(Cmax)was(633±121)μg·L-1,the elimination half-life(T1/2)was(0.72±0.37)h,and the peak time(Tmax)was(0.52±0.16)h.The total clearance rate(CLz)was(1.62±0.26)L·h·kg-1,the area under the curve(AUC0-t)was(946±183)μg·h·L-1,and the mean reten-tion time(MRT0-t)was(1.18±0.17)h.The apparent volume of distribution(Vz)was(1.65±0.86)L·kg-1.The results of tissue distribution study showed that alpha-amanitin was widely distributed in SD rats with the highest concentration in the kidney,followed by the lung,small intestines,stomach wall,LVA blood and liver,but was low in the heart,spleen,testicles and other tissues,and very low in the brain.Alpha-amanitin was absorbed and eliminated quickly,peaked at 40 min in each tissue,and the concen-tration was minimized after 2.5 h.CONCLUSION The absorption and elimination of alpha-amanitin by intraperitoneal injection are rapid in SD rats,and the blood concentration reaches the peak about 31 min after administration,but can not be detected 4 h later.Alpha-amanitin is mainly distributed in the kidney,followed by the tissues and metabolic organs with rich blood flow,such as the lung,small intestines,stomach wall,LVA blood and liver.The content of alpha-amanitin is low in the heart,spleen,testicles and other tissues,and very low in the brain.It is speculated that it may have toxic targeting effect on the kidney and low blood-brain barrier permeability.

Objective:To explore the incidence, diagnosis and treatment of retinopathy of prematurity(ROP) in preterm infants born in the Guangdong Women and Children′s Hospital and transported from other hospital.Method:s Clinical data of 755 premature infants with ROP at Neonatal Intensive Care Unit, Guangdong Women and Children′s Hospital from January 2013 to December 2015 were retrospectively analyzed.There were 239 cases born in the hospital and 516 cases transported from other hospitals.Their gestational age, birth weight, gender, severity of ROP lesion and clinical data were collected and compared.Result:s The birth weight in the group of transported from other hospital was lower than that in the group of born in the hospital[(1 290.64±392.87) g vs.(1 586.21±512.74) g], and the difference was statistically significant( P<0.001). The ROP diagnosis of gestational age in the group of transported from other hospital was higher than that in the group of born in the hospital[(35.53±2.81)weeks vs.(34.51±2.17)weeks], and the difference was statistically significant( P<0.001). On the proportion of severe condition [such as lesion area Ⅰ, aggressive posterior retinopathy of prematurity(AP-ROP) and plus combined lesions], in the group of transported from other hospital was higher than that in the group of born in the hospital, and the differences was statistically significant( P<0.001). In the comparison of the proportion of laser photocoagulation, vitreous injection, combination of the two operations and supplementary laser therapy, in the group of transported from other hospital were higher than those in the group of born in the hospital[60.1%(310/516 cases) vs.20.9%(50/239 cases); 10.9%(56/516 cases) vs.2.5%(6/239 cases); 8.1%(42/516 cases) vs.1.7%(4/239 cases); 4.5%(23/516 cases) vs.1.3%(3/239 cases)], and the differences were statistically significant(all P<0.001). Conclusions:Premature infants with ROP transported from other hospitals have lower birth weight, severe ROP lesions and high surgical intervention rate.Improving ROP screening level in primary hospitals, timely diagnosis and efficient transportation can help to effectively prevent the deterioration of ROP in premature infants and improve their quality of life.

Objective To investigate the risk factors of hypertension patients combined with coronary artery disease(CAD)and analyze the correlation. Methods A total of 258 hospitalized hypertension patients from August 2015 to December 2016 were divided into hypertension group(n = 124)and hypertension combined with CAD group(n = 134),according to the results of coronary angiography. The general data,platelet parame-ters,blood lipid level and renal function indexes of all objects were recorded. The differences of all indexes between 2 groups were compared,and the correlation between these indexes and CAD in hypertensive patients was analyzed. Results(1)More smokers and older patients were found in hypertension combined with CAD group;the values of mean platelet volume(MPV),platelet distribution width,creatinine,serum uric acid were higher, but HDL-C level was lower and all the differences were statistically significant(P < 0.05).(2)Multivariable logistic regression analysis demonstrated that age,smoking history,MPV,serum uric acid and low level of HDL-C were independent hazardous factors of CAD in hypertensive patients[OR = 1.062,95% CI:1.032 ~ 1.093;OR =2.048,95% CI:1.078~3.893;OR=2.737,95% CI:1.193~6.278;OR=1.006,95% CI:1.001~1.010;OR=0.280,95% CI:0.115~0.681(all,P<0.05)].(3)Pearson correlation analysis showed that MPV was positively correlated with serum uric acid(r = 0.17,P < 0.05). Conclusions Age,smoking history,MPV,serum uric acid and lower HDL-C value are risk factors of CAD in hypertensive patients and active detection and prevention will benefit reducing the risk for the occurrence of CAD in hypertensive patients.

The direct negative impact of the transcriptional activity of one component on the second one in c/s is referred to as transcriptional interference (TI).U6 is a type Ⅲ RNA polymerase Ⅲ promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi.In the design and construction of viral vectors,multiple transcription units may be arranged in close proximity in a space-limited vector.Determining if U6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies.In this research,we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units.We arranged U6 promoter driving.shRNA expression and UbiC promoter in two promoter arrangements.In primary macrophages,we found U6 promoter activity was inhibited by UbiC promoter when in the divergent arrangement but not in tandem.In contrast,PKG promoter had no such negative impact.Instead of enhancing U6 promoter activity,CMV enhancer had significant negative impact on U6 promoter activity in the presence of UbiC promoter.Our results indicate that U6 promoter activity can be affected by TI in a proximal promoter-specific and arrangement-dependent manner.

Objective To study the expression and distribution of TIMP 1 and TIMP 2 in liver tissue of cirrhosis patient and to investigate the roles and pathogenesis of TIMP 1 and TIMP 2 in liver cirrhosis. Methods TIMP 1 and TIMP 2 proteins and mRNA were detected with immunohistochemistry and in situ hybridization methods using monoclonal antibodies and cDNA probes. Results mRNA and proteins of TIMP 1 and TIMP 2 were detected in all the liver tissues from 40 liver cirrhosis patients, all in cytoplasm but not nucleus. TIMP 1 and TIMP 2 were found co exist in all samples, while TIMP 1 concentration was higher. Conclusions mRNA and protein of TIMP 1 and TIMP 2 are found in all the cirrhosis patient samples. Liver TIMP 1 and TIMP 2 concentrations increase with the progression of liver cirrhosis, decrease the degradation of extracellular matrix proteins, resulting in the initiation and the development of liver fibrosis and liver cirrhosis.

Objective To observe the inhibition of asON phosphorothioate to the TIMP-1 gene and protein expression in the liver tissue of immune- induced hepatic fibrosis rats. Methods According to the analysis of modulator, structure protein, encoding sequence of TIMP-1 genome, we designed four different groups of asONs. These asONs were injected into the hepatic fibrosis rat models through coccygeal vein. The results were observed by RT-PCR, immunohistochemistry and in situ hybridization with collagen Ⅰ、Ⅲ, special staining of collagen fiber, electron microscope. Results The asON phosphorothioate of TIMP-1 could be expressed in vivo, and could block the TIMP-1 gene and protein expression in the liver of immune- induced hepatic fibrosis rats on the level of mRNA, which could promote the degradation of collagen Ⅰ、Ⅲ(P

An experimental immunity hepatic fibrosis rat model was prepared by means of immunologic assault with human serum albumin, and normal rats served as a control group. The immunohistochemistry methods and in situ hybridization were respectively used to detect TIMP 2 mRNA and related antigens in the liver,and to investigate the localization and expression of TIMP 2 in the liver of both normal and experimental hepatic fibrosis rats. The results showed that TIMP 2 mRNA and related antigens in the livers of experimental group were expressed in myofibroblasts and fibroblasts, especially in the portal area and fibrous septum. The positive signal was located in the cytoplasm, but not in the nucleus. On the other hand, there was a high level of expression of TIMP 2 in the liver of the experimental group.It is suggested that in the process of hepatic fibrosis, fibroblasts and myofibroblasts are the major cells expressing TIMP 2. The severer the hepatic fibrosis in the injured liver is, the higher the levels of TIMP 2 related antigens and gene expression are.