Enhancement of recombinant human interleukin-22 production by fusing with human serum albumin and supplementing N-acetylcysteine in Pichia Pastoris.

Interleukin-22 (IL-22) plays an important role in the treatment of organ failure, which can induce anti-apoptotic and proliferative signaling pathways; Nevertheless, the practical utilization of IL-22 is hindered by the restricted efficacy of its production. Pichia pastoris presents a viable platform for both industrial and pharmaceutical applications. In this study, we successfully generated a fusion protein consisting of truncated human serum albumin and human IL-22 (HSA-hIL-22) using P. pastoris, and examined the impact of antioxidants on HSA-hIL-22 production. We have achieved the production of HSA-hIL-22 in the culture medium at a yield of approximately 2.25 mg/ml. Moreover, 0-40 mM ascorbic acid supplementation did not significantly affect HSA-hIL-22 production or the growth rate of the recombinant strain. However, 80 mM ascorbic acid treatment had a detrimental effect on the expression of HSA-hIL-22. In addition, 5-10 mM N-acetyl-l-cysteine (NAC) resulted in an increase of HSA-hIL-22 production, accompanied by a reduction in the growth rate of the recombinant strain. Conversely, 20-80 mM NAC supplementation inhibited the growth of the recombinant strains and reduced intact HSA-hIL-22 production. However, neither NAC nor ascorbic acid exhibited any effect on superoxide dismutase (SOD) and malondialdehyde (MDA) levels, except that NAC increased GSH content. Furthermore, our findings indicate that recombinant HSA-hIL-22, which demonstrated the ability to stimulate the proliferation of HepG2 cells, possesses bioactivity. In addition, NAC did not affect HSA-hIL-22 bioactivity. In conclusion, our study demonstrates that NAC supplementation can enhance the secretion of functional HSA-hIL-22 proteins produced in P. pastoris without compromising their activity.

Application of attenuation coefficient in the assessment of hepatic involvement in children and adolescents with Wilson's disease.

BACKGROUND: To investigate whether the attenuation coefficient (ATT) can be used as a noninvasive index to assess liver involvement in children and adolescents with Wilson's disease (WD). METHODS: Children and adolescents diagnosed with WD were retrospectively collected from the First Affiliated Hospital of the Anhui University of Traditional Chinese Medicine between May 2022 and August 2022. The findings on ATT, Shear Wave Measurement (SWM), AST to platelet ratio index (APRI), and fibrosis 4 (FIB-4) score were obtained. The liver involvement of WD was classified into 3 groups based on serum levels of collagen type IV (CIV), hyaluronic acid (HA), laminin (LN) and precollagen type III N-terminal peptide (PIIINP): (1) Group1 (n = 25), no abnormalities in CIV, HA, LN and PIIINP; (2) Group2 (n = 19), elevation of 1 or 2 indexes in CIV, HA, LN, and PIIINP; Group3 (n = 18), elevation of 3 or 4 indicators in CIV, HA, LN, and PIIINP. The levels of ATT, SWM, APRI and FIB-4 were compared between the 3 groups; and correlation of ATT with SWM and triglyceride (TG) was performed using Spearman's correlation analysis. The Receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of ATT alone and its combination with SWM, APRI, and FIB-4 in children and adolescents with WD. RESULTS: A total of 62 children and adolescents with WD were retrospectively retrieved. ATT levels were significantly different in intergroup comparisons (P < 0.001). The ROC curve showed that the area under the curve (AUC) for the diagnosis of hepatic steatosis using ATT was 0.714, 0.712 and 0.867 in Group 1 versus Group 2, Group 2 versus Group 3, and Group 1 versus Group 3, respectively; the sensitivity for the diagnosis of hepatic steatosis in Group 1 versus Group 2 was 89.47% with the cutoff value of ATT of 0.73 dB/cm/MHz. No significant correlation found between ATT and TG (ρ = 0.154, P = 0.231). Compared to ATT alone, the combination of ATT with APRI and FIB-4 or the combination of ATT with SWM, APRI, and FIB-4 showed a better diagnostic efficacy in Group 1 versus Group 2 (both P = 0.038). CONCLUSION: ATT could be used as a non-invasive index for the evaluation of liver steatosis in children and adolescents with WD, with a good clinical applicative value. Furthermore, ATT in combination with APRI, FIB-4, and SWM might have better diagnostic efficacy than ATT alone.

Acupoint Catgut Embedding as Adjunctive Therapy for Patients With Gallstones.

AIMS OF THIS STUDY: A randomized clinical trial was undertaken to investigate the efficacy of acupoint catgut embedding (ACE) as adjunctive therapy to tauroursodeoxycholic acid (TUDCA) therapy on gallbladder emptying and clinical symptoms in patients with gallstone disease. MATERIALS AND METHODS: Between August 2018 and January 2019, 70 patients with gallstones in our hospital were enrolled in this prospective clinical trial. All the patients were randomly divided into the ACE group (ACE+TUDCA treatment for 8 wk) and the Sham group (Sham ACE+TUDCA treatment for 8 wk). In the ACE group, all the patients were nightly given ACE every 2 weeks, and in 2 groups, every patient took TUDCA 500 mg at bedtime. The parameters about gallbladder emptying were detected by ultrasound before and after the treatment, and the clinical symptom scores were recorded at the same time points. RESULTS: A total of 63 patients with gallstone disease were included in our study, with 33 patients in the ACE group and 30 patients in the Sham group. In the ACE group, the empty volume (EV) and gallbladder ejection fraction (GBEF) were improved after treatment (P<0.05). Almost every symptom score (except symptom 7, P=0.15) and total score were decreased (P<0.05). In the Sham group, the symptom 1, 2, 4, 5 scores, and total score were significantly decreased (P<0.05). Moreover, the residual volume in the ACE group was significantly lower than in the Sham group (P=0.008). The EV and GBEF in the ACE group were higher than that in the Sham group (P<0.05). The score of symptom 6 in the ACE group was lower than that in the Sham group (P=0.008). CONCLUSION: ACE therapy could more effectively improve the gallbladder emptying with a shorter treatment course. Therefore, ACE+TUDCA therapy might be a time-saving treatment for gallstones.

Dietary plant sterols prevented cholesterol gallstone formation in mice.

Cholesterol gallstone disease is a common global condition. This study investigated the role of plant sterols (PS) in the prevention of gallstone formation and the underlying mechanisms. Adult male mice were fed a lithogenic diet (LD) alone or supplemented with PS (LD-ps), phospholipids (LD-pl) or both PS and phospholipids (LD-ps/pl) for 8 weeks. Incidences of gallstone formation were compared among the groups. Lipids in the bile, liver and serum were analyzed. The expression of genes involved in cholesterol absorption, transport and metabolism in the liver and small intestine was determined. The incidences of gallstone formation were 100% (10/10), 20% (2/10), 100% (10/10) and 40% (4/10) in the LD, LD-ps, LD-pl and LD-ps/pl groups, respectively. Serum cholesterol and intestinal cholesterol absorption were decreased in PS-supplemented mice. The expression of genes related to cholesterol transport and metabolism in the liver was down-regulated by dietary PS. PS supplementation decreased Niemann-Pick C1-like 1 expression in the small intestine and reduced intestinal cholesterol absorption. Our results demonstrated that PS could inhibit intestinal cholesterol absorption and thus prevent cholesterol gallstone formation.

Effect and related mechanism of Yinchenhao decoction on mice with lithogenic diet-induced cholelithiasis.

The aim of the present study was to investigate the effects and the underlying mechanisms of Yinchenhao Decoction (YCHD), a traditional Chinese medicine formulation, on C57BL/6 mice with lithogenic diet (LD)-induced cholelithiasis. The condition of cholelithiasis was evaluated using a six-level criteria. Levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) in the serum and liver tissue were measured using enzyme colorimetry. Concentrations of TC, phospholipids (PL) and total bile acids (TBA) in the bile were measured to calculate the cholesterol saturation index. Liver histopathology was microscopically observed and mRNA expression levels of ABCG5, ABCG8, SRBI, ABCB4, ABCB11 and NPC1L1 involved in cholesterol metabolism were measured using reverse transcription-quantitative PCR. The results showed that feeding mice the LD induced cholelithiasis, along with abnormal serum biochemical indices and imbalances in biliary cholesterol homeostasis. Increased ALT and ALP levels in the serum and ALT, ALP, TC and LDL-C levels in the serum and liver indicated the existence of hepatocyte injury, which were consistent with the pathological changes. YCHD treatment ameliorated the serum and hepatic biochemical abnormalities and adjusted the biliary imbalance. In addition, elevated expression of ATP-binding cassette subfamily G member 5/8, scavenger receptor class B type I and Niemann-Pick C1 Like 1 in the liver and small intestine were observed at the onset of cholelithiasis but were reversed by YCHD. Taken together, results from the present study suggest that YCHD ameliorated LD-induced cholelithiasis mice, which may be caused by improvements in biliary cholesterol supersaturation and regulation of cholesterol metabolism.

Herring egg phosphopeptides as calcium carriers for improving calcium absorption and bone microarchitecture in vivo.

Phosphorylation may enhance the functional properties of proteins/peptides. Herring egg phosphopeptides (HEPPs) have been found to be more effective than the non-phosphorylated variant in calcium-binding activities due to the introduced phosphate groups. However, whether HEPPs as calcium carriers will be superior to herring egg peptides (HEPs) in improving calcium bioavailability in vivo, for the equivalent calcium intake prerequisite, remains to be clarified. This study aimed to evaluate the effect of HEPPs-calcium complex and HEPs-calcium complex on calcium absorption and bioavailability in calcium-deficient mice. Results showed that the remarkably lower calcium absorption and bone calcium deposition induced by long-term calcium deficiency were accompanied by deterioration of the trabecular bone microarchitecture (P < 0.05). The HEPPs-Ca supplements significantly improved the apparent calcium absorption, increased the serum calcium level, decreased the alkaline phosphatase activity, strengthened the bone biomechanical property, and increased bone volume/tissue volume (BV/TV) and trabecular number (Tb·N) in calcium-deficient mice (P < 0.05), as determined by micro-computed tomography (micro-CT) assay. The effect of HEPPs-Ca on calcium absorption and bioavailability was comparable to that of CPPs-Ca, but better than that of HEPs-Ca and CaCO3. This study brings new insights into the potential of HEPPs as an alternative to CPPs for use in calcium supplements.

Effect and Mechanism of ShiZhiFang on Uric Acid Metabolism in Hyperuricemic Rats.

OBJECTIVE: To explore the effect and mechanism of ShiZhiFang on uric acid metabolism. METHODS: 40 rats were divided into normal group, model group, ShiZhiFang group, and benzbromarone group. The hyperuricemic rat model was induced by yeast gavage at 15 g/kg and potassium oxonate intraperitoneal injection at 600 mg/kg for two weeks. During the next two weeks, ShiZhiFang group rats were given ShiZhiFang by gavage, and benzbromarone group rats were given benzbromarone by gavage. The serum uric acid, creatinine, blood urea nitrogen, XOD activity, urinary uric acid, urinary ß2-MG, and histopathological changes were observed in the rats of each group after treatment. RESULTS: The hyperuricemic model was established successfully and did not show the increase of serum creatinine and blood urea nitrogen. Compared with the model group, the serum uric acid, serum XOD activity, and urinary ß2-MG were significantly decreased (p < 0.05), and 24 h urinary uric acid excretion was significantly decreased (p < 0.01) in ShiZhiFang group, whereas the two treatment groups were of no statistical significant in above indicators (p > 0.05); renal histopathology showed that the lesions in two treatment groups were reduced compared to the model groups. The gene and protein expression of uric acid anion transporters rOAT1 and rOAT3 in the kidney was significantly higher than that in model group (p < 0.01). CONCLUSION: The model is suitable for the study of primary hyperuricemia. The mechanisms of ShiZhiFang on uric acid metabolism in hyperuricemic rats may be involved in reducing the activity of serum XOD and promoting the transcription and expression of rOAT1 and rOAT3 in the kidney.

[An establishment of theoretical structure of PRO questionnaire in treating chronic liver disease by Chinese medicine].

By reviewing research contents of patient-reported outcome (PRO) and discussing Chinese medicine (CM) theories related to chronic liver disease (CLD), we have followed international PRO questionnaire development specification, combined CM theories such as uniformed spirit and body, correspondence between human and the universe, yin in property and yang in function of Gan, and seven emotions, and constructed theoretical structure of PRO questionnaire of treating CLD, including four major areas as physiology, psychology, independence, and society and nature. Of them, the physiological field contained six aspects such as blood deficiency, yin deficiency, bleeding, disorder of qi movement, improper transformation and transportation of Pi-Wei, and abnormal biliary excretion. The psychological field contained two aspects: Gan-related emotions and general disease related emotions. The independence field contained two aspects: daily life and study and work. The field of society and nature contains three aspects: social relations, social environment, and natural adaptability.

Microscopy and bioinformatic analyses of lipid metabolism implicate a sporophytic signaling network supporting pollen development in Arabidopsis.

The Arabidopsis sporophytic tapetum undergoes a programmed degeneration process to secrete lipid and other materials to support pollen development. However, the molecular mechanism regulating the degeneration process is unknown. To gain insight into this molecular mechanism, we first determined that the most critical period for tapetal secretion to support pollen development is from the vacuolate microspore stage to the early binucleate pollen stage. We then analyzed the expression of enzymes responsible for lipid biosynthesis and degradation with available in-silico data. The genes for these enzymes that are expressed in the stamen but not in the concurrent uninucleate microspore and binucleate pollen are of particular interest, as they presumably hold the clues to unique molecular processes in the sporophytic tissues compared to the gametophytic tissue. No gene for lipid biosynthesis but a single gene encoding a patatin-like protein likely for lipid mobilization was identified based on the selection criterion. A search for genes co-expressed with this gene identified additional genes encoding typical signal transduction components such as a leucine-rich repeat receptor kinase, an extra-large G-protein, other protein kinases, and transcription factors. In addition, proteases, cell wall degradation enzymes, and other proteins were also identified. These proteins thus may be components of a signaling network leading to degradation of a broad range of cellular components. Since a broad range of degradation activities is expected to occur only in the tapetal degeneration process at this stage in the stamen, it is further hypothesized that the signaling network acts in the tapetal degeneration process.

Isolation, sequence analysis, and expression studies of florally expressed cDNAs in Arabidopsis.

Molecular genetics has identified dozens of genes that regulate flower development in Arabidopsis. However, the complexity of flower development suggests that many other genes are yet to be uncovered. To identify floral genes that are expressed at low levels in the flower, we have sequenced 1587 cDNA fragments from a subtractive floral cDNA library. A total of 1222 unique genes represented by these ESTs are distributed on all five chromosomes with similar frequencies as all predicted genes in the genome. Among these, 17 genes were shown to be expressed anywhere for the first time because they were not found in previous EST and full-length cDNA datasets. Furthermore, 724 of the genes revealed by this library were not definitively shown to be expressed in the flower by previous floral EST datasets. In addition, 49 transcriptional regulators, 31 protein kinases, 12 zinc-finger proteins and other signaling proteins were found to be present in floral buds. Moreover, the EST sequences likely extended the transcribed regions of 26 previously annotated genes, and may have uncovered several previously unrecognized genes. To obtain additional clues about possible gene function, we hybridized cDNA microarray with probes derived from wild-type Arabidopsis rosette leaves and floral buds. We estimated that over 50% of genes were expressed at levels lower than 1/30 of the highest detectable signal intensity, indicating that many floral genes are expressed at low levels. Furthermore, 97 genes were found to be expressed at a higher level in the flower than the leaf by the Significance Analysis of Microarray (SAM) method with a 1.0% false discovery rate (FDR). Further RT-PCR analyses of selected genes support the microarray results. We suggest that the genes encoding putative regulatory proteins and at least some proteins with currently unknown functions might play important roles during flowering.