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.

Irreversible destruction of bronchi and alveoli can lead to multiple incurable lung diseases. Identifying lung stem/progenitor cells with regenerative capacity and utilizing them to reconstruct functional tissue is one of the biggest hopes to reverse the damage and cure such diseases. Here we showed that a rare population of SOX9 basal cells (BCs) located at airway epithelium rugae can regenerate adult human lung. Human SOX9 BCs can be readily isolated by bronchoscopic brushing and indefinitely expanded in feeder-free condition. Expanded human SOX9 BCs can give rise to alveolar and bronchiolar epithelium after being transplanted into injured mouse lung, with air-blood exchange system reconstructed and recipient's lung function improved. Manipulation of lung microenvironment with Pirfenidone to suppress TGF-β signaling could further boost the transplantation efficiency. Moreover, we conducted the first autologous SOX9 BCs transplantation clinical trial in two bronchiectasis patients. Lung tissue repair and pulmonary function enhancement was observed in patients 3-12 months after cell transplantation. Altogether our current work indicated that functional adult human lung structure can be reconstituted by orthotopic transplantation of tissue-specific stem/progenitor cells, which could be translated into a mature regenerative therapeutic strategy in near future.
Bronchiectasis ; genetics ; metabolism ; Humans ; Pulmonary Alveoli ; cytology ; metabolism ; SOX9 Transcription Factor ; genetics ; metabolism ; Stem Cell Transplantation ; methods ; Stem Cells ; cytology ; metabolism

Objective To investigate the expression of TIPE2 in splenic lymphocytes derived from the mouse model of cattle collagen Ⅱ-induced rheumatoid arthritis (CIA) and from peripheral blood of patients with rheumatoid arthritis (RA) for its role in the pathogenesis of RA.Methods The CIA mouse model was established by immunization of DBA-1/J mice with cattle collagen Ⅱ.Peripheral blood lymphocytes were collected from RA patients and healthy donors.The TIPE2 mRNA and protein expression in splenocytes of CIA and control mice were measured by semi-quantitative RT-PCR and Western blot,respectively.In addition,fluorescence quantitative RT-PCR and Western blot were used to determine the TIPE2 mRNA and protein expression in peripheral blood lymphocytes derived from patients with RA and healthy donors.Results Both mRNA and protein expression of TIPE2 were higher in splenocytes of CIA mice and lymphocytes of peripheral blood from RA patients compared with corresponding controls.Furthermore,the mRNA and protein expression of TIPE2 increased significantly in patients with active RA.TIPE2 expression were positively correlated with DAS28 scores (P＜0.001).Conclusion Our results suggest that TIPE2 plays a role in the RA pathogenesis.The level of TIPE2 may also indicate the severity of rheumatoid arthritis.

OBJECTIVETo compare the expressions of lysyl oxidase (LOX) and matrix metalloproteinases-2 (MMP-2) in gastric cancer and pericancerous tissues, in gastric cancers with and without lymph node metastasis, and to analyze the effects of LOX and MMP-2 on tumor invasion and metastasis.

METHODSGastric cancer and pericancerous tissues were collected from 46 patients who underwent surgery. Levels of LOX and MMP-2 mRNA were detected by RT-PCR. Protein abundance of LOX and MMP-2 was examined using Western blot.

RESULTSExpressions of LOX and MMP-2 mRNA, and protein in 46 gastric cancers were significantly higher than that in 46 pericancerous tissues. In gastric cancer with lymph node metastasis, the levels of LOX and MMP-2 mRNA and protein were higher than those in gastric cancers without lymph node metastasis (P < 0.05). In the groups of gastric cancer with lymph node metastasis, expression of LOX was positively correlated with MMP-2 protein expression (P < 0.01).

CONCLUSIONSExpressions of LOX and MMP-2 in gastric cancer tissues are significantly higher than that in pericancerous tissues. The expressions of LOX and MMP-2 in gastric cancer with lymph node metastasis are higher than that in gastric cancer without lymph node metastasis. Expressions of LOX and MMP-2 are positively correlated. The results suggest that LOX and MMP-2 may promote the growth and metastasis of gastric cancer.

Adenocarcinoma ; metabolism ; pathology ; surgery ; Biomarkers, Tumor ; metabolism ; Female ; Gastrectomy ; Humans ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Middle Aged ; Neoplasm Invasiveness ; Protein-Lysine 6-Oxidase ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Stomach ; metabolism ; surgery ; Stomach Neoplasms ; metabolism ; pathology ; surgery

We retrospectively collected the ultrasound imaging data of 152 patients with cirrhosis, the gallbladder wall thickness (GBWT) and portal hemodynamics parameters (portal vein diameter, portal vein mean flow velocity, portal vein blood flow) were recorded,and SPSS 11.0 software was used to analyze the correlation between them. The results revealed that there was good correlation between portal vein diameter and the degree of gallbladder wall thickening (r 0.944, P < 0.05). Portal vein mean velocity and portal vein blood flow were both negatively correlated with gallbladder wall thickness (r = -0.939 or r = -0.950, respectively; both P < or = 0.005). These indicate that gallbladder wall thickening is closely related to hemodynamic parameters. It is feasible to predict the degree of portal hypertension through the observation of gallbladder wall thickening in patients with liver cirrhosis.
Adult ; Aged ; Aged, 80 and over ; Female ; Gallbladder ; pathology ; Hemodynamics ; Humans ; Liver Cirrhosis ; pathology ; physiopathology ; Male ; Middle Aged ; Portal Vein ; physiopathology