Imperatorin ameliorates ferroptotic cell death, inflammation, and renal fibrosis in a unilateral ureteral obstruction mouse model.

BACKGROUND: Imperatorin is a naturally occurring furocoumarin derivative found in traditional Chinese medicine Angelica dahurica for its anticancer, antihypertensive, and antidiabetic properties. Chronic kidney disease (CKD) is a global health issue, characterized by a high prevalence, significant morbidity and mortality, and a range of related complications. OBJECTIVE: This study aims to investigate the protective effects of imperatorin treatment and the specific underlying mechanisms in progressive CKD. METHODS: Imperatorin was orally administrated for 14 consecutive days to mice with unilateral ureteral obstruction (UUO) to investigate the renal pathological alternations, pro-inflammatory mediators, antioxidant response, and ferroptotic death signaling. Imperatorin was also tested in the erastin-induced injury of renal proximal tubular cells (NRK-52E). Cell viability, ferroptosis protein markers, erastin-induced oxidative stress, and lipid peroxidation were assessed. RESULTS: In vivo, imperatorin treatment alleviated kidney histology alternations and attenuated the protein expression of fibrotic markers. Furthermore, imperatorin administration reduced inflammatory cell infiltration, and alleviated the oxidative stress burden by downregulating protein markers such as catalase, superoxide dismutase 2 (SOD-2), NADPH oxidase 4 (NOX-4), and thioredoxin reductase 1 (Trxr-1). It also mitigated ferroptosis markers such as glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11/cystine transporter (SLC7A11/xCT), and transferrin receptor 1 (TFR-1), and attenuated renal cell apoptosis. In vitro, imperatorin treatment effectively decreased erastin-induced feroptotic cell death, restored the antioxidant enzyme levels, and mitigated lipid peroxidation as well as the expression of ferroptosis-related markers (XCT, GPX4, and p-p53) in a dose-dependent manner. CONCLUSION: Our finding demonstrated for the first time, that imperatorin treatment holds therapeutic potential in a UUO mouse model of CKD and inhibits the erastin-induced oxidative stress, ferroptosis, and subsequent lipid peroxidation in vitro. This highlights the potential of imperatorin as a future therapeutic target for ferroptosis to improve the progression of CKD.

Wogonin ameliorates ER stress-associated inflammatory response, apoptotic death and renal fibrosis in a unilateral ureteral obstruction mouse model.

Wogonin, a vital bioactive compound extracted from the medicinal plant, Scutellaria baicalensis, has been wildly used for its potential in mitigating the progression of chronic diseases. Chronic kidney disease (CKD) represents a significant global health challenge due to its high prevalence, morbidity and mortality rates, and associated complications. This study aimed to assess the potential of wogonin in attenuating renal fibrosis and to elucidate the underlying molecular mechanisms using a unilateral ureteral obstruction (UUO) mouse model as a CKD mimic. Male mice, 8 weeks old, underwent orally administrated of either 50 mg/kg/day of wogonin or positive control of 5 mg/kg/day candesartan following UUO surgery. NRK52E cells were exposed to tumor growth factors-beta (TGF-ß) to evaluate the anti-fibrotic effects of wogonin. The results demonstrated that wogonin treatment effectively attenuated TGF-ß-induced fibrosis markers in NRK-52E cells. Additionally, administration of wogonin significantly improved histopathological alterations and downregulated the expression of pro-fibrotic factors (Fibronectin, α-smooth muscle actin, Collagen IV, E-cadherin, and TGF-ß), oxidative stress markers (Catalase, superoxide dismutase 2, NADPH oxidase 4, and thioredoxin reductase 1), inflammatory molecules (Cyclooxygenase-2 and TNF-α), and the infiltration of neutrophils and macrophages in UUO mice. Furthermore, wogonin treatment mitigated endoplasmic reticulum (ER) stress-associated molecular markers (GRP78, GRP94, ATF4, CHOP, and the caspase cascade) and suppressed apoptosis. The findings indicate that wogonin treatment ameliorates key fibrotic aspects of CKD by attenuating ER stress-related apoptosis, inflammation, and oxidative stress, suggesting its potential as a future therapeutic target.

TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.

Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.

Establishment of a human cell line with a surface display system for screening and optimizing Na+-taurocholate cotransporting polypeptide-binding peptides.

One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2-48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A-F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex.

Identification of STAU1 as a regulator of HBV replication by TurboID-based proximity labeling.

The core promoter (CP) of hepatitis B virus (HBV) is critical for HBV replication by controlling the transcription of pregenomic RNA (pgRNA). Host factors regulating the activity of the CP can be identified by different methods. Biotin-based proximity labeling, a powerful method with the capability to capture weak or dynamic interactions, has not yet been used to map proteins interacting with the CP. Here, we established a strategy, based on the newly evolved promiscuous enzyme TurboID, for interrogating host factors regulating the activity of HBV CP. Using this strategy, we identified STAU1 as an important factor involved in the regulation of HBV CP. Mechanistically, STAU1 indirectly binds to CP mediated by TARDBP, and recruits the SAGA transcription coactivator complex to the CP to upregulate its activity. Moreover, STAU1 binds to HBx and enhances the level of HBx by stabilizing it in a ubiquitin-independent manner.

Acute and Long-Term Effects of Mechanotherapy on the Outcome After an Achilles Repair: A Prospective Cohort Study With Historical Controls.

OBJECTIVES: To evaluate the effects of vibration on Achilles' tendon microcirculation and characteristics following surgical repair of Achilles' tendon rupture. DESIGN: Cohort study with historical controls. SETTING: A university institute. PARTICIPANTS: Participants (N=32), including 19 (16 men, 3 women; median [range] age: 43.0 [25.0-57.0] years) and 13 (10 men, 3 women; 44.00 [29.0-60.0] years) in the vibration (application to the ball of the foot, 30Hz, 2mm amplitude, 4kg pressure, and self-administration) and control groups, respectively, who underwent unilateral Achilles' tendon repairs were recruited. INTERVENTION: A 4-week vibration intervention in the vibration group. MAIN OUTCOME MEASUREMENTS: The tendon microcirculation was measured after the first session of vibration. The participants were evaluated repeatedly with bilateral follow-up measurements of tendon stiffness, 3 functional outcome tests, and a questionnaire survey. RESULTS: Acute effects of the vibration were observed immediately after the 5-minute vibration (P≤.001). Lower total hemoglobin and oxygen saturation were respectively observed (P=.043) in the repaired legs 3 and 6 months postsurgery in the vibration group as compared with the control group. The vibration group also showed greater tendon stiffness, heel raising height and hopping distance 3 or 6 months postoperation in both the repaired and noninjured legs (all P<.05). The microcirculatory characteristics 2 months postoperation were correlated with the outcomes at 6 months postoperation. CONCLUSIONS: Differences in microcirculatory characteristics and better rehabilitation outcomes were observed in the legs with an Achilles repair that underwent the early vibration intervention.

SERPINA3 Silencing Inhibits the Migration, Invasion, and Liver Metastasis of Colon Cancer Cells.

OBJECTIVE: To investigate the impact of SERPINA3 on the migration, invasion, and liver metastasis of colon cancer cells. METHODS: Immunohistochemical staining was conducted to determine SERPINA3 expression in the cancer and adjacent normal tissues of 131 patients suffering from colon cancer. In vitro experiment, colon cancer cells with low (HT-29P), intermediate (KM-12C), and high (HT-29LMM, KM-12L4) metastatic potential were obtained to examine SERPINA3 expression levels. Besides, quantitative real-time PCR (qRT-PCR) and Western Blot were performed to detect SERPINA3 expression in HT-29LMM and KM-12L4 cells transfected with SERPINA3 siRNA; Wound-healing and Transwell assays to measure cell migration and invasion, respectively; and ELISA to detect MMP-2 and MMP-9 levels. In vivo experiment, mice with liver metastasis of colon cancer were established to observe the effect of SERPINA3 silencing on liver metastasis. Immunohistochemical assay was applied to evaluate the expressions of Serpina3, Mmp-2, Mmp-9, and proliferating cell nuclear antigen (Pcna) in liver metastasis tissues. RESULTS: SERPINA3 in colon cancer tissues was higher than in adjacent normal tissues, which was associated with patients' clinicopathological features. Besides, SERPINA3 expression showed a rising trend in low, intermediate, and high metastatic potential colon cancer cells. After KM-12L4 and HT-29LMM cells transfected with SERPINA3 siRNA, the migration and invasive ability of cells, as well as the expression levels of MMP-2 and MMP-9 were all decreased. Moreover, SERPINA3 siRNA could not only reduce live metastasis of mice, but also down-regulate the expression of Mmp-2 and Mmp-9 in liver metastasis tissues. CONCLUSION: SERPINA3 silencing could inhibit the migration, invasion, and liver metastasis of colon cancer cells.

Neural stem cell transplantation improves spatial learning and memory via neuronal regeneration in amyloid-ß precursor protein/presenilin 1/tau triple transgenic mice.

Neural stem cell (NSC) transplantation has recently become a main research target for Alzheimer's disease (AD) treatment. In the present study, we transplanted NSCs from C57BL/6 mice into the hippocampus in the 12-month-old triple transgenic model of AD (3 × Tg) and determined whether NSC transplantation can alleviate impairments in spatial learning and memory via neuronal regeneration in AD mice. Two months after transplantation, Morris water maze tests suggested that spatial learning and memory in the 3 × Tg mice receiving NSCs was significantly improved compared to 3 × Tg mice not receiving NSCs. Furthermore, quantification of Nissl staining revealed that the number of neurons in the hippocampus of 3 × Tg mice receiving NSCs was significantly greater than that in 3 × Tg mice not receiving NSCs, indicating that new neurons were generated. These results may demonstrate that NSC transplantation can improve spatial learning and memory via neuronal regeneration in amyloid-ß precursor protein/presenilin 1/tau 3 × Tg mice.

An efficient lipase-catalyzed enantioselective hydrolysis of (R,S)-azolides derived from N-protected proline, pipecolic acid, and nipecotic acid.

In the Candida antarctica lipase B-catalyzed hydrolysis of (R,S)-azolides derived from (R,S)-N-protected proline in water-saturated methyl tert-butyl ether (MTBE), high enzyme activity with excellent enantioselectivity (V (S) V (R) (-1) > 100) for (R,S)-N-Cbz-proline 1,2,4-triazolide (1) and (R,S)-N-Cbz-proline 4-bromopyrazolide (2) was exploited in comparison with their corresponding methyl ester analog (3). Changing of the substrate structure, water content, solvent, and temperature was found to have profound influences on the lipase performance. On the basis of enzyme activity and enantioselectivity and solvent boiling point, the best reaction condition of using 1 as the substrate in water-saturated MTBE at 45 °C was selected and further employed for the successful resolution of (R,S)-N-Cbz-pipecolic 1,2,4-triazolide (5) and (R,S)-N-Boc-nipecotic 1,2,4-triazolide (9). Moreover, more than 89.1 % recovery of remained (R)-1 is obtainable in five cycles of enzyme reusage, when pH 7 phosphate buffers were employed as the extract at 4 °C.

Kinetic and thermodynamic analysis for lipase-catalyzed hydrolytic resolution of (R,S)-alcohols though their azolyl carbamates.

A new approach to the lipase-catalyzed hydrolytic resolution of (R,S)-azolyl carbamates for obtaining chiral azolyl carbamates and alcohol is described. With (R,S)-1-phenylethyl azolyl carbamates as the model substrates, the best reaction condition of using (R,S)-1-phenylethyl 4-bromopyrazole carbamate (1) as the substrate in water-saturated diisopropyl ether at 45 °C is selected. The kinetic constants, and hence enantiomeric ratio of 124, are then estimated from the kinetic analysis by considering the alcohol inhibition effect, with which theoretical time-course conversions for both enantiomers are numerically solved and agree with the experimental data. The thermodynamic parameters -ΔΔH and -ΔΔS satisfying a linear enthalpy-entropy compensation relationship of -ΔΔS = -38.84 + 3.29(-ΔΔH) are further estimated. An extension of the resolution platform to (R,S)-4-bromopyrazole carbamates derived from other (R,S)-alcohols (4, 5, 7) is also addressed.

(R,S)-2-chlorophenoxyl pyrazolides as novel substrates for improving lipase-catalyzed hydrolytic resolution.

The best reaction condition of Candida antartica lipase B as biocatalyst, 3-(2-pyridyl)pyrazole as leaving azole, and water-saturated methyl t-butyl ether as reaction medium at 45°C were first selected for performing the hydrolytic resolution of (R,S)-2-(4-chlorophenoxyl) azolides (1-4). In comparison with the kinetic resolution of (R,S)-2-phenylpropionyl 3-(2-pyridyl)pyrazolide or (R,S)-α-methoxyphenylacetyl 3-(2-pyridyl)pyrazolide at the same reaction condition, excellent enantioselectivity with more than two order-of-magnitudes higher activity for each enantiomer was obtained. The resolution was then extended to other (R,S)-3-(2-pyridyl)pyrazolides (5-7) containing 2-chloro, 3-chloro, or 2,4-dichloro substituent, giving good (E > 48) to excellent (E > 100) enantioselectivity. The thermodynamic analysis for 1, 2, and 4-7 demonstrates profound effects of the acyl or leaving moiety on varying enthalpic and entropic contributions to the difference of Gibbs free energies. A thorough kinetic analysis further indicates that on the basis of 6, the excellent enantiomeric ratio for 4 and 7 is due to the higher reactivity of (S)-4 and lower reactivity of (R)-7, respectively.

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization.

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)-mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 43.6 of (R,S)-ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM-87 from the producer) to 16.7 mmol/h g and 867 of (R,S)-2-methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)-2-hydroxycarboxylic acid esters, giving improvements of the enzyme performance from V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)-2-methoxyethyl 3-chloromandelate (16) for the immobilized enzyme, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 8.27 of (R,S)-ethyl 4-chloromandelate (10) for free SNSM-87 to 33.5 mmol/h g and 123 of (R,S)-methyl 4-chloromandelate (14) for the immobilized enzyme, as well as V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 7.94 of (R,S)-ethyl 3-phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)-2-methoxyethyl 3-phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach.

Hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media: implication for rate-limiting formation or breakdown of tetrahedral intermediates in acylation step.

A thermally stable esterase (SNSM-87) from Klebsiella oxytoca is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure-enantioselectivity correlations and substrate partitioning in the aqueous phase. With (R,S)-2-chloromandelates as the model substrates, an expanded Michaelis-Menten mechanism for the rate-limiting acylation step is adopted for the kinetic analysis. The Brønsted slope of 25.7 for the fast-reacting (S)-2-chloromandelates containing a difficult leaving alcohol moiety, as well as that of 4.13 for the slow-reacting (R)-2-chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is rate-limiting. However, the rate-limiting step shifts to the formation of tetrahedral intermediates for the (S)-2-chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate.