The reason for the amelioration of N-methyl-N-nitrosourea-induced retinitis pigmentosa in rats by hydrogen-rich saline

AIM：To investigate the effects of hydrogen-rich saline（HRS）on microglia activation and Sirtuin type 1（Sirt1）in rats with N-methyl-N-nitrosourea（MNU）-induced retinitis pigmentosa（RP）.METHODS：Rats were divided into norm（N）group,model（M）group and HRS（H）group.Rats in M and H groups were given saline and HRS respectively prior to and after administration of MNU.At one day（d1）and d3 afterwards,electroretinogram and histological examination were performed to confirm the effects of HRS on retinal function and structure of MNU-induced RP.Immunofluorescence staining of anti-ionized calcium-binding adapter molecule 1（Iba1）,a maker of microglia cells,was performed,with quantitative real-time polymerase chain reaction（qRT-PCR）for its m RNA quantification.Moreover,Sirt1 m RNA and protein expression in the retinas were detected by Western blot and qRT-PCR.RESULTS：HRS preserved the retinal function and mitigated the reduction of photoreceptor degeneration in MNU-treated retinas.The presence of microglia cells was somewhat more obvious in H group than that in M group at d1.HRS suppressed the further activation of microglia cells,with the number of microglia cells less than that of M group at d3.Results of qRT-PCR of Iba1 were consistent with those of immunofluorescence staining,with the m RNA expression of Iba1 in H group more intensive than that of M group at d1（P〈0.05）,while less than that of M group at d3（P〈0.05）.Furthermore,the Sirt1 m RNA and protein expression decreased after MNU administration,while HRS mitigated the MNU-induced downregulation of Sirt1.CONCLUSION：HRS can effectively keep microglia activation induced by MNU to an appropriate extent,while upregulate Sirt1 in MNU-induced RP.

Autophagy dysregulation mediates the damage of high glucose to retinal pigment epithelium cells

AIM:To observe the role and mechanism of autophagy in retinal pigment epithelial cell(RPE)damaged by high glucose,so as to offer a new idea for the treatment of diabetic retinopathy(DR).METHODS:ARPE-19,a human RPE cell line cultured in vitro was divided into the normal control(NC),autophagy inhibitor 3-methyladenine(3-MA),high-glucose(HG),and HG+3-MA groups.Cell viability was detected by CCK-8 assay and the apoptosis rate was measured by flow cytometry.The protein expressions of apoptosis markers,including Bax,Bcl-2,and Caspase-3,as well as autophagy marker including microtubule-related protein 1 light chain 3(LC3),p62,and mechanistic target of rapamycin(m TOR)were detected by Western blotting.Autophagic flux was detected by transfection with Ad-m Cherry-GFP-LC3 B.RESULTS:Under high glucose conditions,the viability of ARPE-19 was decreased,and the apoptosis rate increased,the protein expressions of Bax,Caspase-3,and LC3-II/LC3-I were all increased and the expressions of Bcl-2,p62 and p-m TOR decreased,and autophagic flux was increased compared with that of the controls.Treatment with 3-MA reversed all these changes caused by high glucose.CONCLUSION:The current study demonstrates the mechanisms of cell damage of ARPE-19 through high glucose/m TOR/autophagy/apoptosis pathway,and new strategies for DR may be developed based on autophagy regulation to manage cell death of RPE cells.

Tetanic contraction induces enhancement of fatigability and sarcomeric damage in atrophic skeletal muscle and its underlying molecular mechanisms

Muscle unloading due to long-term exposure of weightlessness or simulated weightlessness causes atrophy, loss of functional capacity, impaired locomotor coordination, and decreased resistance to fatigue in the antigravity muscles of the lower limbs. Besides reducing astronauts' mobility in space and on returning to a gravity environment, the molecular mechanisms for the adaptation of skeletal muscle to unloading also play an important medical role in conditions such as disuse and paralysis. The tail-suspended rat model was used to simulate the effects of weightlessness on skeletal muscles and to induce muscle unloading in the rat hindlimb. Our series studies have shown that the maximum of twitch tension and the twitch duration decreased significantly in the atrophic soleus muscles, the maximal tension of high-frequency tetanic contraction was significantly reduced in 2-week unloaded soleus muscles, however, the fatigability of highfrequency tetanic contraction increased after one week of unloading. The maximal isometric tension of intermittent tetanic contraction at optimal stimulating frequency did not alter in 1-and 2-week unloaded soleus, but significantly decreased in 4-week unloaded soleus. The 1-week unloaded soleus, but not extensor digitorum longus(EDL), was more susceptible to fatigue during intermittent tetanic contraction than the synchronous controls. The changes in K+ channel characteristics may increase the fatigability during high-frequency tetanic contraction in atrophic soleus muscles. High fatigability of intermittent tetanic contraction may be involved in enhanced activity of sarcoplasmic reticulum Ca2+-ATPase(SERCA) and switching from slow to fast isoform of myosin heavy chain, tropomyosin, troponin I and T subunit in atrophic soleus muscles. Unloaded soleus muscle also showed a decreased protein level of neuronal nitric oxide synthase(nNOS), and the reduction in nNOS-derived NO increased frequency of calcium sparks and elevated intracellular resting Ca2+ concentration([Ca2+]i) in unloaded soleus muscles. High [Ca2+]i activated calpain-1 which induced a higher degradation of desmin. Desmin degradation may loose connections between adjacent myofibrils and further misaligned Z-disc during repeated tetanic contractions. Passive stretch in unloaded muscle could preserve the stability of sarcoplasmic reticulum Ca2+ release channels by means of keeping nNOS activity, and decrease the enhanced protein level and activity of calpain to control levels in unloaded soleus muscles. Therefore, passive stretch restored normal appearance of Z-disc and resisted in part atrophy of unloaded soleus muscles. The above results indicate that enhanced fatigability of high-frequency tetanic contraction is associated to the alteration in K+ channel characteristics, and elevated SERCA activity and slow to fast transition of myosin heavy chain(MHC) isoforms increases fatigability of intermittent tetanic contraction in atrophic soleus muscle. The sarcomeric damage induced by tetanic contraction can be retarded by stretch in atrophic soleus muscles.

HuR-mediated nucleocytoplasmic translocation of HOTAIR relieves its inhibition of osteogenic differentiation and promotes bone formation

Bone marrow mesenchymal stem cell(BMSC)osteogenic differentiation and osteoblast function play critical roles in bone formation,which is a highly regulated process.Long noncoding RNAs(lncRNAs)perform diverse functions in a variety of biological processes,including BMSC osteogenic differentiation.Although several studies have reported that HOX transcript antisense RNA(HOTAIR)is involved in BMSC osteogenic differentiation,its effect on bone formation in vivo remains unclear.Here,by constructing transgenic mice with BMSC(Prx1-HOTAIR)-and osteoblast(Bglap-HOTAIR)-specific overexpression of HOTAIR,we found that Prx1-HOTAIR and Bglap-HOTAIR transgenic mice show different bone phenotypes in vivo.Specifically,Prx1-HOTAIR mice showed delayed bone formation,while Bglap-HOTAIR mice showed increased bone formation.HOTAIR inhibits BMSC osteogenic differentiation but promotes osteoblast function in vitro.Furthermore,we identified that HOTAIR is mainly located in the nucleus of BMSCs and in the cytoplasm of osteoblasts.HOTAIR displays a nucleocytoplasmic translocation pattern during BMSC osteogenic differentiation.We first identified that the RNA-binding protein human antigen R(HuR)is responsible for HOTAIR nucleocytoplasmic translocation.HOTAIR is essential for osteoblast function,and cytoplasmic HOTAIR binds to miR-214 and acts as a ceRNA to increase Atf4 protein levels and osteoblast function.BglapHOTAIR mice,but not Prx1-HOTAIR mice,showed alleviation of bone loss induced by unloading.This study reveals the importance of temporal and spatial regulation of HOTAIR in BMSC osteogenic differentiation and bone formation,which provides new insights into precise regulation as a target for bone loss.

Effect of rotary motion on Fos protein expression in the vestibular-related nucleus population in a mouse model of rapid retinal degeneration

BACKGROUND： Previous studies on integration mechanisms of visual and vestibular information in the central nervous system have focused on the vestibular system. Due to the lack of an appropriate animal model, few studies have addressed the visual system with regard to visual and vestibular information. OBJECTIVE： To investigate Fos protein expression differences of vestibular-related nucleus populations in a mouse model of rapid retinal degeneration and normal wild-type Kunming mice following rotary motion, and to verify integration regions of visual and vestibular information in the central nervous system. DESIGN, TIME AND SETTING： A randomized, controlled in vitro study was performed at the Key Laboratory of Aerospace Medicine of Ministry of Education, China from March 2008 to February 2009. MATERIALS： A rotary stimulation device was re-fit to an electric, rotating chair produced by the School of Aerospace Medicine, the Fourth Military Medical University. METHODS： A total of 12 rapid retinal degeneration mice and 12 normal wild-type male Kunming mice were randomly assigned to experimental and control subgroups, respectively （n = 6）. Mice in the experimental group were exposed to rotary motion at a speed of 180°/s, 3 minutes per cycle, in an alternating clockwise/counterclockwise movement. Mice in the control group were not exposed to rotary motion. MAIN OUTCOME MEASURES： Differences in the number of Fos-positive neurons were determined in the vestibular nucleus, prepositus hypoglossal nucleus, inferior olive subnucleus beta, Kooy cap of the inferior olive medial nucleus, and the flocculus and paraflocculus of the cerebellum in rapid retinal degeneration mice and normal wild-type Kunming mice. RESULTS： The number of Fos-positive neurons was reduced in the prepositus hypoglossal nucleus and the Kooy cap of the inferior olive medial nucleus in the rapid retinal degeneration mice following 30 minutes of rotary motion in the experimental group, compared with the normal wild-type Kunming mice （P 〈 0.01）. There was no significant difference in Fos protein expression in the vestibular nucleus, inferior olive subnucleus beta, and the flocculus and paraflocculus of the cerebellum between the rapid retinal degeneration mice and normal wild-type Kunming mice. CONCLUSION： Visual information affected neuronal activation in the prepositus hypoglossal nucleus and the Kooy cap of the inferior olive medial nucleus in mice following rotary motion. The prepositus hypoglossal nucleus and the dorsal cap of Kooy of inferior olive medial nucleus were shown to be key integration regions of visual information and vestibular information in the central nervous system.

Microgravity-induced cardiovascular deconditioning:mechanisms and countermeasures

It has been demonstrated that individuals exposed to actual or simulated microgravity often experience cardiovascular dysfunctions when returning to Earth.The underlying mechanisms of orthostatic intolerance and countermeasure treatment are still poorly understood.In this paper,the progress in the mechanism of cardiovascular deconditioning from the view of vascular remodeling,increased venous compliance in the lower limbs,cellular proliferation and differentiation,and cell signaling pathway was reviewed.Meanwhile,an overview of the countermeasures including exercise,lower body negative pressure,thigh cuffs,traditional Chinese herb medicine and artificial gravity was presented.

The mechanosensitive lncRNA Neat1 promotes osteoblast function through paraspeckle-dependent Smurf1 mRNA retention

Mechanical stimulation plays an important role in bone remodeling. Exercise-induced mechanical loading enhances bone strength,whereas mechanical unloading leads to bone loss. Increasing evidence has demonstrated that long noncoding RNAs(lnc RNAs) play key roles in diverse biological, physiological and pathological contexts. However, the roles of lnc RNAs in mechanotransduction and their relationships with bone formation remain unknown. In this study, we screened mechanosensing lnc RNAs in osteoblasts and identified Neat1, the most clearly decreased lnc RNA under simulated microgravity. Of note, not only Neat1 expression but also the specific paraspeckle structure formed by Neat1 was sensitive to different mechanical stimulations, which were closely associated with osteoblast function. Paraspeckles exhibited small punctate aggregates under simulated microgravity and elongated prolate or larger irregular structures under mechanical loading. Neat1 knockout mice displayed disrupted bone formation, impaired bone structure and strength, and reduced bone mass. Neat1 deficiency in osteoblasts reduced the response of osteoblasts to mechanical stimulation. In vivo, Neat1 knockout in mice weakened the bone phenotypes in response to mechanical loading and hindlimb unloading stimulation. Mechanistically, paraspeckles promoted nuclear retention of E3 ubiquitin ligase Smurf1 m RNA and downregulation of their translation, thus inhibiting ubiquitination-mediated degradation of the osteoblast master transcription factor Runx2, a Smurf1 target. Our study revealed that Neat1 plays an essential role in osteoblast function under mechanical stimulation, which provides a paradigm for the function of the lnc RNA-assembled structure in response to mechanical stimulation and offers a therapeutic strategy for long-term spaceflight-or bedrest-induced bone loss and age-related osteoporosis.

Mitochondrial Oxidative Stress Enhances Vasoconstriction by Altering Calcium Homeostasis in Cerebrovascular Smooth Muscle Cells under Simulated Microgravity

Objective Exposure to microgravity results in postflight cardiovascular deconditioning in astronauts.Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process.To elucidate the mechanism for this condition,we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted(HU)rat cerebral arteries.Methods Three-week HU was used to simulate microgravity in rats.The contractile responses to vasoconstrictors,mitochondrial fission/fusion,Ca^(2+) distribution,inositol 1,4,5-trisphosphate receptor(IP3 R)abundance,and the activities of voltage-gated K+channels(KV)and Ca^(2+)-activated K+channels(BKCa)were examined in rat cerebral vascular smooth muscle cells(VSMCs).Results An increase of cytoplasmic Ca^(2+) and a decrease of mitochondrial/sarcoplasmic reticulum(SR)Ca^(2+) were observed in HU rat cerebral VSMCs.The abundance of fusion proteins(mitofusin 1/2[MFN1/2])and fission proteins(dynamin-related protein 1[DRP1]and fission-mitochondrial 1[FIS1])was significantly downregulated and upregulated,respectively in HU rat cerebral VSMCs.The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats,and IP3 R protein/mRNA levels were significantly upregulated.The current densities and open probabilities of KV and BKCa decreased and increased,respectively.Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1.It also decreased IP3 R expression levels and restored the activities of the KV and BKCa channels.MitoTEMPO restored the Ca^(2+) distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries.Conclusion The present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.

Drug discovery of sclerostin inhibitors

Sclerostin, a protein secreted from osteocytes, negatively regulates the WNT signaling pathway by binding to the LRP5/6 co-receptors and further inhibits bone formation and promotes bone resorption. Sclerostin contributes to musculoskeletal system-related diseases, making it a promising therapeutic target for the treatment of WNT-related bone diseases. Additionally, emerging evidence indicates that sclerostin contributes to the development of cancers, obesity, and diabetes, suggesting that it may be a promising therapeutic target for these diseases. Notably, cardiovascular diseases are related to the protective role of sclerostin. In this review, we summarize three distinct types of inhibitors targeting sclerostin, monoclonal antibodies, aptamers, and small-molecule inhibitors, from which monoclonal antibodies have been developed. As the first-in-class sclerostin inhibitor approved by the U.S. FDA,the monoclonal antibody romosozumab has demonstrated excellent effectiveness in the treatment of postmenopausal osteoporosis;however, it conferred high cardiovascular risk in clinical trials. Furthermore,romosozumab could only be administered by injection, which may cause compliance issues for patients who prefer oral therapy. Considering these above safety and compliance concerns, we therefore present relevant discussion and offer perspectives on the development of next-generation sclerostin inhibitors by following several ways, such as concomitant medication, artificial intelligence-based strategy, druggable modification, and bispecific inhibitors strategy.

Advanced nerve regeneration enabled by neural conformal electronic stimulators enhancing mitochondrial transport

Addressing peripheral nerve defects remains a significant challenge in regenerative neurobiology.Autograftsemerged as the gold-standard management,however,are hindered by limited availability and potential neuromaformation.Numerous recent studies report the potential of wireless electronic system for nerve defects repair.Unfortunately,few has met clinical needs for inadequate electrode precision,poor nerve entrapment andinsufficient bioactivity of the matrix material.Herein,we present an advanced wireless electrical nerve stimulator,based on water-responsive self-curling silk membrane with excellent bioabsorbable and biocompatibleproperties.We constructed a unique bilayer structure with an oriented pre-stretched inner layer and a generalsilk membrane as outer layer.After wetting,the simultaneous contraction of inner layer and expansion of outerlayer achieved controllable super-contraction from 2D flat surface to 3D structural reconfiguration.It enablesshape-adaptive wrapping to cover around nerves,overcomes the technical obstacle of preparing electrodes on theinner wall of the conduit,and prevents electrode breakage caused by material expansion in water.The use of forkcapacitor-like metal interface increases the contact points between the metal and the regenerating nerve,solvingthe challenge of inefficient and rough electrical stimulation methods in the past.Newly developed electronicstimulator is effective in restoring 10 mm rat sciatic nerve defects comparable to autologous grafts.The underlyingmechanism involves that electric stimulation enhances anterograde mitochondrial transport to matchenergy demands.This newly introduced device thereby demonstrated the potential as a viable and efficaciousalternative to autografts for enhancing peripheral nerve repair and functional recovery.

RIPK3 promotes hantaviral replication by restricting JAK-STAT signaling without triggering necroptosis

Hantaan virus(HTNV)is a rodent-borne virus that causes hemorrhagic fever with renal syndrome(HFRS),resulting in a high mortality rate of 15%.Interferons(IFNs)play a critical role in the anti-hantaviral immune response,and IFN pretreatment efficiently restricts HTNV infection by triggering the expression of a series of IFNstimulated genes(ISGs)through the Janus kinase-signal transducer and activator of transcription 1(JAK-STAT)pathway.However,the tremendous amount of IFNs produced during late infection could not restrain HTNV replication,and the mechanism remains unclear.Here,we demonstrated that receptor-interacting protein kinase 3(RIPK3),a crucial molecule that mediates necroptosis,was activated by HTNV and contributed to hantavirus evasion of IFN responses by inhibiting STAT1 phosphorylation.RNA-seq analysis revealed the upregulation of multiple cell death-related genes after HTNV infection,with RIPK3 identified as a key modulator of viral replication.RIPK3 ablation significantly enhanced ISGs expression and restrained HTNV replication,without affecting the expression of pattern recognition receptors(PRRs)or the production of type I IFNs.Conversely,exogenously expressed RIPK3 compromised the host's antiviral response and facilitated HTNV replication.RIPK3^(-/-)mice also maintained a robust ability to clear HTNV with enhanced innate immune responses.Mechanistically,we found that RIPK3 could bind STAT1 and inhibit STAT1 phosphorylation dependent on the protein kinase domain(PKD)of RIPK3 but not its kinase activity.Overall,these observations demonstrated a noncanonical function of RIPK3 during viral infection and have elucidated a novel host innate immunity evasion strategy utilized by HTNV.

Multi-Model Ensemble Deep Learning Method to Diagnose COVID-19 Using Chest Computed Tomography Images

Deep learning based analyses of computed tomography(CT)images contribute to automated diagnosis of COVID-19,and ensemble learning may commonly provide a better solution.Here,we proposed an ensemble learning method that integrates several component neural networks to jointly diagnose COVID-19.Two ensemble strategies are considered:the output scores of all component models that are combined with the weights adjusted adaptively by cost function back propagation;voting strategy.A database containing 8347 CT slices of COVID-19,common pneumonia and normal subjects was used as training and testing sets.Results show that the novel method can reach a high accuracy of 99.37%(recall:0.9981;precision:0.9893),with an increase of about 7% in comparison to single-component models.And the average test accuracy is 95.62%(recall:0.9587;precision:0.9559),with a corresponding increase of 5.2%.Compared with several latest deep learning models on the identical test set,our method made an accuracy improvement up to 10.88%.The proposed method may be a promising solution for the diagnosis of COVID-19.