[Preliminary Study on the Effect of Silencing Nucleostemin Com- bined with Rapamycin on Autophagy and Apoptosis of HL-60 Cells].

OBJECTIVE: To investigate the effects of knocking down nucleostemin ( NS) combined with rapamycin (RAPA) on autophagy and apoptosis in HL-60 cells , and to explore its role in HL-60 cells . METHODS: The expression of NS protein was detected using Western blot , after transfection of HL-60 cells was achieved by the recombinant lentviral vector NS -RNAi-GV248 . Flow cytometry was used to detect changes in cells apoptosis after NS silencing/ rapamycin for 24 , 48 hours , and the expressions of NS , LC3 , p62 , BCL-2 and Bax proteins in cells were detected by Western blot. RESULTS: The expression of NS in HL-60 cells was successfully down-regulated by recombinant lentiviral vector. After treatment with rapamycin for 24 and 48 h , the apoptosis rate of cells in each group increased (P < 0.05) , and the apoptosis was more obvious at 48 hours . Compared with the NS silencing group or rapamycin group , after treated with NS down-regulation combined with rapamycin for 48 hours , the apoptosis of HL-60 cells was significantly increased ( P < 0.05 ) , LC3 -II/LC3 -I ratio was significantly increased ( P < 0.05 ) , p62 protein expression was significantly decreased (P < 0.05) , and BCL-2/Bax ratio was significantly decreased ( P < 0.05) . CONCLUSION: NS down-regulation combined with rapamycin can enhance the apoptosis and autophagy of HL-60 cells , and the induction of apoptosis of HL-60 cells may be related to the expression of BCL-2 and Bax proteins .

[Significance of anti-Jo-1 antibody's clinical stratification in idiopathic inflammatory myopathy and disease spectrum].

OBJECTIVE: To investigate the significance of anti-histidyl tRNA synthetase (Jo-1) antibody in idiopathic inflammatory myopathies (IIM) and its diseases spectrum. METHODS: We enrolled all the patients who were tested positive for anti-Jo-1 antibody by immunoblotting in Peking University People's Hospital between 2016 and 2022. And the patients diagnosed with anti-synthetase antibody syndrome (ASS) with negative serum anti-Jo-1 antibody were enrolled as controls. We analyzed the basic information, clinical characteristics, and various inflammatory and immunological indicators of the patients at the onset of illness. RESULTS: A total of 165 patients with positive anti-Jo-1 antibody were enrolled in this study. Among them, 80.5% were diagnosed with connective tissue disease. And 57.6% (95/165) were diagnosed with IIM, including ASS (84/165, 50.9%), immune-mediated necrotizing myopathy (7/165, 4.2%) and dermatomyositis (4/165, 2.4%). There were 23.0% (38/165) diagnosed with other connective tissue disease, mainly including rheumatoid arthritis (11/165, 6.7%), undifferentiated connective tissue disease (5/165, 3.0%), interstitial pneumonia with autoimmune features (5/165, 3.0%), undifferentiated arthritis (4/165, 2.4%), Sjögren's syndrome (3/165, 1.8%), systemic lupus erythematosus (3/165, 1.8%), systemic vasculitis (3/165, 1.8%), and so on. Other cases included 3 (1.8%) malignant tumor patients, 4 (2.4%) infectious cases and so on. The diagnoses were not clear in 9.1% (15 /165) of the cohort. In the analysis of ASS subgroups, the group with positive serum anti-Jo-1 antibody had a younger age of onset than those with negative serum anti-Jo-1 antibody (49.9 years vs. 55.0 years, P=0.026). Clinical manifestations of arthritis (60.7% vs. 33.3%, P=0.002) and myalgia (47.1% vs. 22.2%, P=0.004) were more common in the ASS patients with positive anti-Jo-1 antibody. With the increase of anti-Jo-1 antibody titer, the incidence of the manifestations of arthritis, mechanic hands, Gottron sign and Raynaud phenomenon increased, and the proportion of abnormal creatine kinase and α-hydroxybutyric dehydrogenase index increased in the ASS patients. The incidence of myalgia and myasthenia were significantly more common in this cohort when anti-Jo-1 antibody-positive ASS patients were positive for one and more myositis specific antibodies/myositis associated autoantibodies (P < 0.05). CONCLUSION: The disease spectrum in patients with positive serum anti-Jo-1 antibody includes a variety of diseases, mainly ASS. And anti-Jo-1 antibody can also be found in many connective tissue diseases, malignant tumor, infection and so on.

[Treadmill exercise alleviates neuropathic pain by regulating mitophagy of the anterior cingulate cortex in rats].

This study aimed to investigate the effect of treadmill exercise on neuropathic pain and to determine whether mitophagy of the anterior cingulate cortex (ACC) contributes to exercise-mediated amelioration of neuropathic pain. Chronic constriction injury of the sciatic nerve (CCI) was used to establish a neuropathic pain model in Sprague-Dawley (SD) rats. Von-Frey filaments were used to assess the mechanical paw withdrawal threshold (PWT), and a thermal radiation meter was used to assess the thermal paw withdrawal latency (PWL) in rats. qPCR was used to evaluate the mRNA levels of Pink1, Parkin, Fundc1, and Bnip3. Western blot was used to evaluate the protein levels of PINK1 and PARKIN. To determine the impact of the mitophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP) on pain behaviors in CCI rats, 24 SD rats were randomly divided into CCI drug control group (CCI+Veh group), CCI+CCCP low-dose group (CCI+CCCP0.25), CCI+CCCP medium-dose group (CCI+CCCP2.5), and CCI+CCCP high-dose group (CCI+CCCP5). Pain behaviors were assessed on 0, 1, 3, 5, and 7 days after modeling. To explore whether exercise regulates pain through mitophagy, 24 SD rats were divided into sham, CCI, and CCI+Exercise (CCI+Exe) groups. The rats in the CCI+Exe group underwent 4-week low-moderate treadmill training one week after modeling. The mechanical pain and thermal pain behaviors of the rats in each group were assessed on 0, 7, 14, 21, and 35 days after modeling. Western blot was used to detect the levels of the mitophagy-related proteins PINK1, PARKIN, LC3 II/LC3 I, and P62 in ACC tissues. Transmission electron microscopy was used to observe the ultrastructure of mitochondrial morphology in the ACC. The results showed that: (1) Compared with the sham group, the pain thresholds of the ipsilateral side of the CCI group decreased significantly (P < 0.001). Meanwhile, the mRNA and protein levels of Pink1 were significantly higher, and those of Parkin were lower in the CCI group (P < 0.05). (2) Compared with the CCI+Veh group, each CCCP-dose group showed higher mechanical and thermal pain thresholds, and the levels of PINK1 and LC3 II/LC3 I were elevated significantly (P < 0.05, P < 0.01). (3) The pain thresholds of the CCI+Exe group increased significantly compared with those of the CCI group after treadmill intervention (P < 0.001, P < 0.01). Compared with the CCI group, the protein levels of PINK1 and P62 were decreased (P < 0.001, P < 0.01), and the protein levels of PARKIN and LC3 II/LC3 I were increased in the CCI+Exe group (P < 0.01, P < 0.05). Rod-shaped mitochondria were observed in the ACC of CCI+Exe group, and there were little mitochondrial fragmentation, swelling, or vacuoles. The results suggest that the mitochondrial PINK1/PARKIN autophagy pathway is blocked in the ACC of neuropathic pain model rats. Treadmill exercise could restore mitochondrial homeostasis and relieve neuropathic pain via the PINK1/PARKIN pathway.

[Correlation of Nucleostemin with Programmed Death-ligand-1 in Multiple Myeloma U266 Cells].

OBJECTIVE: To investigate the correlation of nucleostemin (NS) gene with programmed death ligand-1 (PD-L1) in myeloma cells and the effect of NS expression down-regulation on the apoptosis of multiple myeloma cells, and to evaluate the associations among NS, PD-L1 and biological behavior of MM cells, and the feasibility of both NS and PD-1 as markers reflecting the status of MM cells. METHODS: The NS gene expression in U266 cells was down-regulated by NS-RNAi-GV248 recombinant lentivirus, the real-time PCR was used to detect the mRNA expression of NS, PD-L1 and PI3K/AKT/mTOR. The Western blot and flow cytometry were used to detect the expression of NS and PD-L1. The Annexin V-APC/7-AAD staining method was used to detect the apoptosis of U266 cells before and after knocking out the NS gene. RESULTS: Under the condition of MOI=10, the transfection efficiency was more than 75% by means of the fluorescent microscopy; real-time PCR showed that compared with the negative control group (1.002±0.026), the mRNA expression of NS, PD-L1 and PI3K/AKT/mTOR gene in the transfection group (0.415±0.089) was significantly reduced (P＜0.05). The results of flow cytometry and Western blot showed that the protein expression of PD-L1 was significantly down-regulated after transfection. After down-regulation of NS gene expression, the apoptosis of U266 cells increased (P＜0.05). CONCLUSION: The abnormal high expression of NS and PD-L1 genes exists in U266 cells, moreover, the down-regulation of PD-L1 and the related PISK/AKT/mTOR pathway gene expression appears after down-regulation of NS gene expression, which suggest that the cell biological changes resulted from above-mentioned results, show a synergestic effect on U266 cells.

Efficacy and safety of autologous stem cell transplantation for decompensated liver cirrhosis: A retrospective cohort study.

AIM: To evaluate the long-term efficacy and safety of autologous stem cell transplantation (SCT) for decompensated liver cirrhosis. METHODS: Consecutive patients with decompensated liver cirrhosis were included and assigned into the SCT group and non-transplantation (non-SCT) group according to whether they received SCT treatment. Patients were followed up for ten years. The long-term survival rate and incidence of hepatocellular carcinoma (HCC) were compared between groups. RESULTS: A total of 159 patients were enrolled, including 27 cases in the SCT group and 132 cases in the non-SCT group. The baseline characteristics were significantly different between the two groups. Propensity score matching (PSM) was used to match SCT and non-SCT patients. After PSM, 92 subjects were enrolled in the final analysis, including 23 cases in the SCT group and 69 cases in the non-SCT group. The overall mortality was 73.9% and 55.1%, and the median survival period was 48 and 64 mo, respectively. However, no significant difference was found in the long-term survival rate between the two groups (P > 0.05). In addition, the incidence of HCC was higher in the SCT group than in the non-SCT group (47.8% vs 21.7%, P < 0.05). After adjusting for other covariates, SCT (OR = 3.065, 95%CI: 1.378-6.814) and age (OR = 1.061, 95%CI: 1.021-1.102) were independently correlated with the development of HCC in this decompensated liver cirrhosis cohort. CONCLUSION: Autologous SCT may fail to improve the long-term efficacy and increase the incidence of HCC for decompensated liver cirrhosis. Close monitoring of HCC is strongly recommended in patients undergoing autologous SCT.

[Network pharmacology of flavonoids in Sophora alopecuroides].

The aim of this paper was to investigate the potential pharmacological effect of flavonoids in Sophora alopecuroides by network pharmacology. This study predicted the potential targets of 11 flavonoids of S. alopecuroides with help of reversed pharmacophore matching target recognition service platform (PharmMapper). The pathway information was acquired from DAVID and KEGG databases. Cytoscape software was used to construct the "ingredient-target-pathway" network of flavonoids active components of S. alopecuroides. The flavonoids active components of S. alopecuroides play anti-inflammatory, blood sugar regulating and other pharmacological effects by regulating 62 targets (such as INSR，KDR，MET) and intervening 44 pathways, such as B cell receptor signaling pathway, insulin signaling pathway, neurotrophin signaling pathway, and T cell receptor signaling pathway. In this study, the mechanism of "muti components-multitargets-multiple pathway" of flavonoids was studied. It reflects the multi-components, multi-targets and multiple pathway features of traditional Chinese medicine. Meanwhile, it provides a scientific basis for the elucidation the mechanism of S. alopecuroides as a medicine, and the development and utilization resources of S. alopecuroides.

[Research on network pharmacology of alkaloids in Sophora alopecuroides].

It was aimed at exploring the potential pharmacological effects of alkaloids in Sophora alopecuroides by means of network pharmacology in this study. The main alkaloids in S. alopecuroides were collected for analysis of drug properties, prediction of potential targets and screening of signaling pathways. DAVID analysis tool combined with KEGG database was used to annotate and analyze the signaling pathway. The alkaloids-targets-signaling pathways network was built through Cytoscape software. Results showed that 17 alkaloids in S. alopecuroides involved 49 targets (170 times in all) and 22 important signaling pathways. Three nodes in model of network pharmacology were cross-linked, and the metabolic pathways were coordinated and regulated by each other. It indicated that alkaloids in S. alopecuroides may have therapeutic effect on diseases of cancer, metabolic disorder, endocrine system, digestive system, nervous system and so on.

[Effects of different drying methods on content of bioactive component and antioxidant activity in Lycium ruthenicum].

To compare the appearances, tastes, contents of bioactive components and antioxidant activity of Lyceum ruthenicum under different drying methods, so as to direct its production practice. The folin-phenol colorimetric method, UV, extinction coefficient method and DPPH, as well as fluorescence recovery after photobleaching (FRAP) method to determine the contents of polyphenols, proanthocyanidins, total anthocyanin and antioxidant activity under different drying methods： vacuum freeze drying, low-temperature oven drying and air drying for L. ruthenicum. The results showed that the drying methods had certain effects on its appearances, tastes, contents of bioactive components and antioxidant activity. The appearances and tastes were best after the L. ruthenicum was dried by vacuum freeze drying, with significantly lower moisture than air drying method. The contents of total polyphenols, anthocyanin and proanthocyanidins were highest by air-drying but lowest by low temperature oven drying in L. ruthenicum. The scavenging ability to DPPH was strongest by freeze-drying and lowest by low temperature oven drying, while the antioxidant activity was strongest by air-drying in the FRAT method. In addition, the appearances and tastes were poor in air drying, with higher moisture but highest contents of the three bioactive components. Therefore, the drying methods for L. ruthenicum shall be comprehensively considered.

[Comparative identification studies on wild and cultivated Glycyrrhiza uralensis produced Ningxia based on index components and near-infrared spectroscopy].

This study is to construct a rapid and effective method for identification of wild and cultivated Glycyrrhiza uralensis (hereinafter referred to as Glycyrrhizae Radix et Rhizoma) from Ningxia by comparison of the difference in chromatography identification based on index components and near-infrared spectroscopy identification. HPLC and UV methods were used to determine the content of liquiritin, glycyrrhizate and total flavonoids for 9 wild Glycyrrhizae Radix et Rhizoma and 14 cultivated Glycyrrhizae Radix et Rhizoma samples,and the near-infrared spectroscopy was also,collected. The results illustrated that the chromatography identification based on index components could not identify wild and cultivated Glycyrrhizae Radix et Rhizoma from Ningxia, while near-infrared spectroscopy could quickly and effectively achieve it. It provides an effective method for the growth pattern identification and application of Glycyrrhizae Radix et Rhizoma.

[Comparative studies on HPLC fingerprint and near-infrared spectra of cultivated and reference crude Scutellaria baicalensis].

Scutellaria baicalensis is a common and important medicinal plant in China, facing with reducing sharply in wild resources. To meet the needs in Chinese herbwouls medicine market and clinical application, S. baicalensis has been widely cultivated in Ningxia, Hebei, Shanxi, and Gansu et al. HPLC finger-print and near-infrared were studied in the research to evaluate quality difference of S. baicalensis in four districts. The results showed that the similarity of HPLC finger-print of 12 cultivated S. baicalensis and reference crude herb is more than 0.961, and the other is more than 0.983. On the other hand, paired sample t-test indicated there has no significant difference between the common peaks' area of 12 cultivated S. baicalensis and reference crude herb. It was verified that 12 cultivated S. baicalensis has highly consistency with reference crude herb. On the basis of chromatographic finger-print and near-infrared spectrum, the study applied paired sample t-test to verify analysis results, which could avoid erroneous judgment induced by indefinite threshold values in the similarity of chromatographic finger-print and provide reliable basis for the analysis results. Meanwhile, it also provides a new idea for improving the quality control method of Chinese medicinal materials by comparative study about two comprehensive detection means.

Hyperammonaemia induces hepatic injury with alteration of gene expression profiles.

BACKGROUND: Hyperammonaemia is a serious metabolic disorder commonly observed in patients with hepatic failure. However, it is unknown whether hyperammonaemia has a direct adverse effect on the hepatocytes and thereby serves as both a cause and effect of hepatic failure. AIMS: The purposes were to determine whether hepatic injury can be caused by hyperammonaemia, and if so, screen the key genes involved in hyperammonaemia. METHODS: Hyperammonaemic rats were established via intragastric administration of the ammonium chloride solution. The liver tissues were assessed via biochemistry, histology, immunohistochemistry and microarray analysis. Selected genes were confirmed by quantitative RT-PCR. RESULTS: Administration of the ammonium chloride caused the hyperammonaemia, accompanied with the changes of plasma markers indicating hepatic injury. A pathological assessment demonstrated increased apoptosis and higher level of cyclin D1 and cyclin A in hyperammonaemic rat liver. Microarray was performed on the liver samples and 198 differentially expressed genes were identified in hyperammonaemic rats and validated by quantitative RT-PCR. These genes were associated with many vital functional classes and belonged to different signal transduction pathways. CONCLUSIONS: This study demonstrates that hyperammonaemia can directly induce hepatic injury via the hepatocyte apoptosis. Gene expression profile may provide the possible explanations and mechanisms for the hepatic injury induced by hyperammonaemia.

[Hyperammonemia-induced hepatic injury in rats: characterization of a new animal model].

OBJECTIVE: To establish an accurate new rat model of hyperammonemia-induced liver injury for use in studies of the molecular mechanisms underlying acute liver failure (ALF). METHODS: Twenty-six Sprague-Dawley rats were administered D-galactosamine (400 mg/kg) and endotoxin (50 mug/kg) via intraperitoneal injection to induce ALF and sacrificed at 12 h post-injection (ALF-12 group, n = 10) or 24 h post-injection (ALF-24 group, n = 16). Ten rats administered physiological saline served as the control group. In addition, 20 rats were given serial oral administrations of 10% NH4Cl solution (10 ml/kg, every 8 hrs) to establish the hyperammonemia-induced liver injury model; an additional 20 rats were prepared in parallel to serve as the ALF control group (n = 10; D-galactosamine at 800 mg/kg every 6 d for 30 days) and the physiological saline control group (n = 10). Serum samples were collected from each mouse and used to detect markers of liver function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alpha-fetal protein (AFP), and gamma-glutamyltransferase (GGT), as well as blood ammonia (BA) level and prothrombin time activity (PT-A). Affects on liver histology was assessed by hematoxylin and eosin staining of resected liver tissues, and on apoptosis by TUNEL assay and calculating the apoptotic index (AI). RESULTS: ALF rats showed elevated levels of ALT (1202.51+/-282.00 U/L), AST (1560.14+/-298.98 U/L), and BA (165.9+/-23.6 mumol/L) as early as 6 hrs after model establishment; these levels peaked at 12 hrs after model establishment (ALT: 774.40+/-207.65 U/L; AST: 967.60+/-121.94 U/L; BA: 143.4+/-18.1 mumol/L; P less than 0.05). No significant variations were detected in the levels of AFP (except for the ALF-24 group) or GGT. Liver tissues of the ALF-12 and ALF-24 groups showed large or diffuse hemorrhagic necroses with sinusoidal congestion or spotty bleeding, as well as increased AI. Hyperammonemia-induced liver injury rats showed elevated levels of ALT and BA as early as 6 hrs after model establishment. Similar to the ALF rats, AFP and GGT were unaffected and AI increased. However, in contrast to the ALF rats, the liver tissues of the hyperammonemia-induced liver injury rats showed no signs of hepatocyte swelling, necrosis, or inflammatory cell invasion. CONCLUSION: ALF rats and hyperammonemia-induced liver injury rats have elevated BA and marked hepatocyte necrosis. Given that reducing the level of ammonemia can improve the animal's biochemistry indexes, it is likely that hyperammonemia plays a role in acute liver injury or ALF consequent to repeated injury. The pathogenic mechanisms of repeated injury may involve promotion of hepatocyte apoptosis in conjunction with inhibition of cellular regeneration.

[Optimization of extraction process of total alkaloids from radix of Aconitum flavum using response surface methodology].

OBJECTIVE: To study the technological parameters of the extraction process of total alkaloids from Radix of Aconitum flavum. METHODS: Using response surface methodology (RSM) of three factors and three levels to optimize ethanol heat reflux applied for alkaloids extraction from Radix of Aconitum flavum. RESULTS: A quadratic polynomial mathematical model with good correlation was constructed and employed to the optimization. The optimal extraction conditions were as follows: the extraction time was 1.72 h, the concentration of ethanol was 64.00%, the ratio of ethanol to raw material was 8.18 mL/g, under which the predictive value of the rate of extraction yield of alkaloids was 0.526% and the measured value was 0. 521%, respectively, and there was a good agreement with their predicted values. CONCLUSION: This reveals the good predictability of the mathematical model, the selected process also has a good reproducibility.

[Clinical analysis of 58 cases intraoperative image-guidance in nasal endoscopic surgery].

OBJECTIVE: To evaluate the availability and our experience of intraoperative image-guidance in endoscopic nasal surgery. METHODS: Fifty-eight cases of endoscopic nasal surgery with intraoperative image-guidance were retrospectively reviewed, including 39 cases of chronic sinusitis with or without nasal polyp; 3 cases of necrotizing maxillary sinusitis; 6 cases of sphenoid sinus cyst; 2 cases of nasopharyngeal angiofibroma; 1 case of cranio-nasal meningioma; 1 case of traumatic cerebrospinal rhinorrhea; 1 case of traumatic optic nerve lesion. All patients were operated on with Brain Lab operation imaging navigation system and nasal endoscope. RESULTS: The preoperative preparatory time would take 10-20 minutes, including coordination, head holder localization, conventional instrument registration. In our cases, the localization accuracy between 3-D image landmarks of navigation system and actual anatomical landmarks was less than 1.5 mm. The optic nerve and other anatomical points could be orientated accurately in intraoperative procedures. No complication occurred. CONCLUSIONS: Nasal endoscope combined with image-guidance systems provides accurate anatomical localization of nasal cavity, sinuses and anterior skull base with enlarged operation field. It is possible for surgeons to observe the surrounding important anatomical structures during endoscopic nasal surgery. It could increase the effectiveness and decrease surgical complications, especially in complicated cases.