Novel insights into the protective effects of leonurine against acute kidney injury: Inhibition of ER stress-associated ferroptosis via regulating ATF4/CHOP/ACSL4 pathway.

Acute kidney injury (AKI) is a common and serious global health problem with high risks of mortality and the development of chronic kidney diseases. Leonurine is a unique bioactive component from Leonurus japonicus Houtt. and exerts antioxidant, antiapoptotic or anti-inflammatory properties. This study aimed to explore the benefits of leonurine on AKI and the possible mechanisms involved, with a particular foc on the regulation of ferroptosis and endoplasmic reticulum (ER) stress. Our results showed that leonurine exhibited prominent protective effects against AKI, as evidenced by the amelioration of histopathological alterations and reduction of renal dysfunction. In addition, leonurine significantly suppressed ferroptosis in AKI both in vivo and in vitro by effectively restoring ultrastructural abnormalities in mitochondria, decreasing ASCL4 and 4-HNE levels, scavenging reactive oxygen species (ROS), as well as increasing GPX4 and GSH levels. In parallel, leonurine also markedly mitigated ER stress via down-regulating PERK, eIF-2α, ATF4, CHOP and CHAC1. Further studies suggested that ER stress was closely involved in erastin-induced ferroptosis, and leonurine protected tubular epithelial cells in vitro by inhibiting ER stress-associated ferroptosis via regulating ATF4/CHOP/ASCL4 signalling pathway. Mechanistically, ATF4 silencing in vitro regulated CHOP and ACSL4 expressions, ultimately weakening both ER stress and ferroptosis. Notably, analyses of single-cell RNA sequencing data revealed that ATF4, CHOP and ASCL4 in renal tubular cells were all abnormally upregulated in patients with AKI compared to healthy controls, suggesting their contributions to the pathogenesis of AKI. Altogether, these findings suggest that leonurine alleviates AKI by inhibiting ER stress-associated ferroptosis via regulating ATF4/CHOP/ASCL4 signalling pathway, thus providing novel mechanisms for AKI treatment.

LncRNA FTX ameliorates neuropathic pain by targeting miR-320a in a rat model of chronic constriction injury.

INTRODUCTION: Long non-coding RNAs (lncRNAs) participate in the process of neuropathic pain (NP). Herein, the goal of this research was to examine the roles of lncRNA five prime to XIST (FTX) in influencing chronic constriction injury (CCI)-induced NP. MATERIAL AND METHODS: We have established a rat CCI model to simulate NP in vivo. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect mRNA levels of FTX, microRNA (miR)-320a, and runt-related transcription factor 2 (RUNX2) in the spinal cord. This was followed by subsequent regulation of FTX or miR-320a levels in vivo by intrathecal injection of overexpression FTX or miR-320a mimic lentivirus. The behaviour of rat NP the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of pro-inflammatory and anti-inflammatory factors in the spinal cord tissue. A correlation between FTX and miR-320a, and RUNX2 was validated by luciferase reporter. RESULTS: FTX levels were reduced in CCI rats ( p < 0.05), and miR-320a was a direct target of FTX. Overexpression of FTX typically reduced PWL and PWT as well as neuroinflammation thus alleviating NP ( p < 0.05). However, increasing miR-320a reversed the alleviation of FTX on NP, increased PWL and PWT, and promoted neuroinflammation ( p < 0.05). Additionally, RUNX2, which is a miR-320a target gene, was significantly repressed in CCI rats and its expression was increased by FTX, however, this increase was attenuated by elevated miR-320a ( p < 0.05). CONCLUSIONS: In the CCI-induced NP rat model, FTX attenuates NP and neuroinflammation by regulating the miR-320a/RUNX2 axis. This provides a new vision for NP treatment.

Clerodendranthus spicatus inhibits epithelial-mesenchymal transition of renal tubular cells through the NF-κB/Snail signalling pathway in hyperuricaemia nephropathy.

CONTEXT: Clerodendranthus spicatus Thunb. (Labiatae) (CS), a perennial traditional Chinese medicinal herb that can reduce serum uric acid (sUA) levels and ameliorate renal function is widely used to treat hyperuricaemic nephropathy (HN). OBJECTIVE: To investigate the molecular mechanism of action of CS in HN treatment using in vivo and in vitro experiments. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into control, HN, CS and positive control allopurinol groups. The HN group was intraperitoneally injected with 750 mg/kg oxonic acid potassium (OA), whereas the CS group was injected with OA along with a gavage of CS (low dose 3.125 g/kg, high dose 6.25 g/kg) for five weeks. For in vitro studies, uric acid-treated HK2 cells were used to verify the therapeutic mechanism of CS in HN. RESULTS: HN rats exhibit pathological phenotypes of elevated sUA levels and renal injury. CS significantly improved these symptoms and sUA (p < 0.05) and blood urea nitrogen (p < 0.01) levels, and dramatically improved renal tubular injury in HN rats. The IC50 value of UA (uric acid) in HK2 cells was 826.32 ± 3.55 µg/mL; however, 120 ng/mL CS had no significant cytotoxicity on HK2 cells. In vivo and in vitro studies showed that CS inhibited NF-κB phosphorylation and inhibited α-smooth muscle actin (α-SMA) and vimentin expression while increasing E-cadherin expression, suggesting that CS inhibited the fibrotic process in renal cells, thus protecting renal function. DISCUSSION AND CONCLUSIONS: These findings provide a fundamental understanding of the application of CS in HN treatment to better guide clinical interventions.

Association between sociodemographic status and cardiovascular risk factors burden in community populations: implication for reducing cardiovascular disease burden.

BACKGROUND: We aimed to evaluate the burden of cardiovascular (CV) risk factors in the community populations of Guangdong Province and its association with sociodemographic status (SDS). METHOD: The data were from the community populations of Guangdong Province who have participated in the China PEACE Million Persons Project between 2016 and 2020 (n = 102,358, women 60.5% and mean age 54.3 years). The prevalence of CV risk factors (smoking, drinking, overweight/obesity, hypertension, dyslipidemia and diabetes mellitus) and its association with SDS (age, sex and socioeconomic status [SES]) was evaluated cross-sectionally. RESULTS: The prevalence of overweight/obesity was 48.9%, hypertension 39.9%, dyslipidemia 18.6%, smoking 17.2%, diabetes mellitus 16.1% and drinking 5.3%. Even in young adults (aged 35-44), nearly 60% had at least 1 CV risk factor. Overweight/obesity often coexisted with other risk factors, including smoking, hypertension, dyslipidemia and diabetes mellitus. The proportion of people with no risk factor decreased with increasing age. Women were more likely than men to have no CV risk factor (29.4% vs. 12.7%). People with ≥ high school degree were more likely than those with < high school to have no risk factor (28.5% vs. 20.4%), and farmers were less likely than non-farmers to have no risk factor (20.8% vs. 23.1%). CONCLUSION: The burden of CV risk factors is high and varied by SDS in the community populations of Guangdong Province. Cost-effective and targeted interventions are needed to reduce the burden of CV risk factors at the population level.

Corrigendum: Sanqi Oral Solution Mitigates Proteinuria in Rat Passive Heymann Nephritis and Blocks Podocyte Apoptosis via Nrf2/HO-1 Pathway.

[This corrects the article DOI: 10.3389/fphar.2021.727874.].

Sanqi Oral Solution Mitigates Proteinuria in Rat Passive Heymann Nephritis and Blocks Podocyte Apoptosis via Nrf2/HO-1 Pathway.

Idiopathic membranous nephropathy (IMN) is the most common pathological type in adult nephrotic syndrome where podocyte apoptosis was found to mediate the development of proteinuria. Sanqi oral solution (SQ), an effective Chinese herbal preparation clinically used in treatment of IMN for decades, plays an important role in reducing proteinuria, but the underlying mechanisms have not been fully elucidated yet. The current study tested the hypothesis that SQ directly lessens proteinuria in IMN by reducing podocyte apoptosis. To investigate the effects of SQ, we established the experimental passive Heymann nephritis (PHN) rat model induced by anti-Fx1A antiserum in vivo and doxorubicin hydrochloride (ADR)-injured apoptotic podocyte model in vitro. SQ intervention dramatically reduced the level of proteinuria, together with the rat anti-rabbit IgG antibodies, complement C3, and C5b-9 deposition in glomerulus of PHN rats, accompanied by an elevation of serum albumin. Protein expression of synaptopodin, marker of podocyte injury, restored after SQ administration, whereas the electron microscopic analysis indicated that fusion of foot processes, and the pachynsis of glomerular basement membrane was markedly diminished. Further studies showed that SQ treatment could significantly inhibit podocyte apoptosis in PHN rats and ADR-injured podocytes, and protein levels of Cleaved Caspase-3 or the ratio of Bax/Bcl-2 were significantly decreased with SQ treatment in vivo or in vitro. Moreover, we found that the nuclear factor erythroid 2-related factor-2/heme oxygenase 1 (Nrf2/HO-1) pathway mediated the anti-apoptosis effective of SQ in podocyte. Thus, SQ mitigates podocyte apoptosis and proteinuria in PHN rats via the Nrf2/HO-1 pathway.

Sanqi Oral Solution Ameliorates Renal Ischemia/Reperfusion Injury via Reducing Apoptosis and Enhancing Autophagy: Involvement of ERK/mTOR Pathways.

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI) is a significant health problem with high morbidity and mortality, yet prophylaxis strategies and effective drugs are limited. Sanqi oral solution (SQ) is a formulated medicine widely used in clinical settings to treat various renal diseases via enriching qi and activating blood circulation while its role on I/R-AKI remains unclear. Herein, by establishing rat I/R-AKI models, we intended to investigate the effect of SQ on the prevention of I/R-AKI and explore its underlying mechanisms. We demonstrated that SQ treatment significantly attenuated renal dysfunction of I/R-AKI, alleviated histological damages, inhibited renal apoptosis, and enhanced autophagy. Further investigation proved that SQ could significantly inhibit the activation of ERK and mTOR signaling pathways. Moreover, its renoprotective effect can be abolished by autophagy inhibitor 3-methyladenine (3-MA). Collectively, our results suggest that SQ exerts renoprotective effects on renal I/R injury via reducing apoptosis and enhancing autophagy, which are associated with regulating ERK/mTOR pathways.

Insight into the HIV-1 Vif SOCS-box-ElonginBC interaction.

The HIV-1 viral infectivity factor (Vif) neutralizes cell-encoded antiviral APOBEC3 proteins by recruiting a cellular ElonginB (EloB)/ElonginC (EloC)/Cullin5-containing ubiquitin ligase complex, resulting in APOBEC3 ubiquitination and proteolysis. The suppressors-of-cytokine-signalling-like domain (SOCS-box) of HIV-1 Vif is essential for E3 ligase engagement, and contains a BC box as well as an unusual proline-rich motif. Here, we report the NMR solution structure of the Vif SOCS-ElonginBC (EloBC) complex. In contrast to SOCS-boxes described in other proteins, the HIV-1 Vif SOCS-box contains only one α-helical domain followed by a ß-sheet fold. The SOCS-box of Vif binds primarily to EloC by hydrophobic interactions. The functionally essential proline-rich motif mediates a direct but weak interaction with residues 101-104 of EloB, inducing a conformational change from an unstructured state to a structured state. The structure of the complex and biophysical studies provide detailed insight into the function of Vif's proline-rich motif and reveal novel dynamic information on the Vif-EloBC interaction.

Factors affecting the effective number of tests in genetic association studies: a comparative study of three PCA-based methods.

The number of tested marker becomes numerous in genetic association studies (GAS) and one major challenge is to derive the multiple testing threshold. Some approaches calculating an effective number (M(eff)) of tests in GAS were developed and have been shown to be promising. As yet, there have been no comparisons of their robustness to influencing factors. We evaluated the performance of three principal component analysis (PCA)-based M(eff) estimation formulas (M(eff-C) in Cheverud (2001), M(eff-L) in Li and Ji (2005), and M(eff-G) in Galwey (2009)). Four influencing factors including LD measurements, marker density, population samples and the total number of tested markers were considered. We validated them by the Bonferroni's method and the permutation test with 10 000 random shuffles based on three real data sets. For each factor, M(eff-C) yielded conservative threshold except with D' coefficient, and M(eff-G) would be too liberal compared with the permutation test. Our results indicated that M(eff-L) based on r(2) coefficient achieve close approximation of the permutation threshold. As for a large number of markers, we recommended to use M(eff-L) with r(2) coefficient according to fixed-length separation, as well as fixed-number separation, to obtain accurate estimate of the multiple testing threshold and to save more computational time.

Soluble expression, purification, and characterization of recombinant human flotillin-2 (reggie-1) in Escherichia coli.

Large scale production of recombinant human flotillin-2 (reggie-1) is desirable for structural and biochemical studies. However, as the major lipid rafts specific hydrophobic protein, flotillin-2 was difficult to be expressed as soluble and functional form in prokaryotic system. In this study, we first cloned and expressed human flotillin-2 in Escherichia coli with five different fusion tags: poly-histidine, glutathione S-transferase (GST), thioredoxin (TRX), N-Utilization substance (NusA) and maltose binding protein (MBP). We screened the expression level and solubility of the five flotillin-2 fusion proteins, the best MBP tagged flotillin-2 was then large scale produced. The optimized purification procedure included two steps of chromatography: Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 36.0 mg soluble and functional recombinant flotillin-2 from 1 L of culture medium with purity above 97%. The activity of recombinant flotillin-2 was verified by pull-down assay with flotillin-1, showing that the purified recombinant flotillin-2 can specifically interact with flotillin-1. The circular dichroism (CD) spectroscopy showed that recombinant flotillin-2 had a very stable secondary structure dominated by α-helix, ß-turn and random structure.

A novel method for high-level production of psychrophilic TAB5 alkaline phosphatase.

Heat labile alkaline phosphatases (APs) are widely used in biomedical research for they can easily be heat inactivated once they have done their job. Here we reported a novel method for high-level production of recombinant psychrophilic Antarctic bacterium strain TAB5 alkaline phosphatase (TAP) in Escherichia coli. We synthesized the whole TAP gene according to the synonymous codon choice that is optimal for the E. coli translational system. Then the gene was cloned into pT7 expression vector, expressed in BL21 (DE3) pLysS strain by auto-induction system. The recombinant protein was purified by Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 90.9 mg protein from 16.2 g wet cells in 1L culture medium with the purity over 99%, 340 times as many mg/L (and 21 times the mg/g cells) compared to previous methods. The dephosphorylation activity assay showed that the purified recombinant TAP has similar activity to calf intestinal alkaline phosphatase in room temperature, and it can be totally inactivated by treatment at 60°C for 15 min. Our research provides a novel method for high-level expression, purification and characterization of TAP which is sufficient for high throughput genome analysis and may replace the widely used shrimp AP because of its low cost.

A novel method for high-level production of TEV protease by superfolder GFP tag.

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants of sfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant of sfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. The sfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications.