| BackgroundSarcopenia is an age-related syndrome characterized by progressive and extensive loss of skeletal muscle strength and mass,and the major pathological manifestations of sarcopenia are the decrease in muscle cells and the increase in interstitial fibrous and adipose tissues.Muscle tissue content is reported to gradually decrease from the age of 35 in humans,and sarcopenia affects almost all elderly people.Sarcopenia triggers numerous adverse events,including falls,loss of function,weakness,and even death.In this century,population aging is becoming more and more obvious.Consequently,it is of great significance to explore the the potential mechanisms of and effective intervention targets for sarcopeniaAtrophy of muscle cells is a major histopathological change of sarcopenia.Skeletal muscle cells consist of slow muscle cells(type Ⅰ)and fast muscle cells(type II).As reported,both the size and the number of skeletal muscle cells decrease in the elderly people,and the decline occurs more obviously in fast muscle cells.Although exercise training can partially alleviate the loss of fast muscle cells,it cannot completely prevent it.The decrease of fast muscle cells accounts for the declined capacity to generate explosive force in old people,whereas the retention of slow muscle cells allows the elderly to move slowly.Thus,it is important to alleviate skeletal muscle cell atrophy,especially fast muscle cell atrophy for improving the exercise capacity of old people.Accumulation of interstitial fibrous tissue is another major histopathological change of sarcopenia.Interstitial fibrosis is clearly observed in aging skeletal muscles,and the increase in fibrous tissue leads to enhanced muscle stiffness,which restricts muscle stretching and contraction,and thereby lowers the exercise capacity of the elderly people.Moreover,the deposition of fibrous tissue might interfere with the interactions between satellite cells and surrounding cells and thus lead to impaired muscle regeneration.Thus,alleviating skeletal muscle fibrosis is expected to improve skeletal muscle function.Atrophy of muscle cells and accumulation of interstitial fibrous tissue are both major histopathological changes of sarcopenia and important causes for declined muscle function and decreased exercise capacity in old people.How to reduce muscle cell atrophy and inhibit interstitial fibrosis is the key to alleviate sarcopenia in the elderly population.TRB3 might serve as a bridge between skeletal muscle cell atrophy,interstitial fibrosis,and impaired exercise capacity in old people.TRB3 was previously reported to exhibit age-related expression and play a vital role in cell proliferation,differentiation,and fibrosis.It has been demonstrated that TRB3 caused muscle cell atrophy and functional recession by negatively modulating protein turnover in the condition of food deprivation and could inhibit the myogenic differentiation of C2C12 cells.TRB3 was also found to induce interstitial fibrosis in various diseases such as systemic sclerosis,diabetic nephropathy,and diabetic cardiomyopathy.Therefore,it is reasonable to speculate that altered TRB3 expression might play a vital role in age-related skeletal muscle cell atrophy and interstitial fibrosisHerein,we investigated the effects of TRB3 on skeletal muscle cell atrophy and fibrosis in naturally aging wild-type(WT)and TRB3 knockout mice.Objectives1.To investigate whether TRB3 expression is associated with the occurrence and development of sarcopenia in aging mice;2.To investigate the possible mechanisms by which TRB3 knockout alleviated sarcopenia in aging mice.Methods1.AnimalsFour-week-old male wild type(WT)and TRB3 knockout(TRB3-/-)C57/BL6 mice were randomized into young and old groups:WT young group,WT old group,TRB3-/-young group,and TRB3-/-old group.The young-and old-group mice were raised until they were 3 and 18 months old,respectively.2.Assessment of exercise capacityExercise capacity was tested at the end of the experiment.Forelimb grip strength test:The dynamometer was placed horizontally,the mice were held by their tails and trained to grasp the dynamometer with their forepaws.Then the mice were gently pulled backward until they released the dynamometer.Forelimb grip strength of each mouse was tested thrice,and the three measured values were recorded and averaged.Hanging grid test:A 45×45 cm grid was placed on a 55-cm-high frame and a 5-cm-thick cushion was placed under the grid.We placed each mouse at the center of the grid and then turned the grid upside down with the mouse head declining first.Hanging time was recorded as the time until the mice fell.Each mouse was tested thrice with a>30-min interval between tests,and the hanging time was recorded and averaged.Exhaustive running test:The mice were placed on the treadmill.The running was started at 13 m/min with a slope angle of 0°,and then the speed and slope angle were increased by 2 m/min and 2° every 3 min until they reached 39 m/min and 14°,respectively.When the mouse being tested did not return to the track for>20 s and concomitantly exhibited a markedly diminished response to external stimuli,the exhaustive running time was recorded.3.Assessment of muscle functionMice were anesthetized,the extensor digitorum longus(EDL)muscle was exposed,and the distal tendon was tied with a surgical suture and dissected at the distal end of the knot.Next,the muscle was separated up to the proximal muscle attachment site and the other end of the suture was tied to a force transducer and maintained at a low base tension.The muscle was connected to an isolated pulse stimulator by a platinum wire and stimulated at 20 V/cm.Stimulation with a 5-ms square wave was used while the base tension was gradually increased until the optimal base tension was discovered.With the optimal base tension maintained,5-ms pulses at 0.2 Hz were applied thrice to determine the maximal isometric twitch force,and 5-ms pulses at 100 Hz for 300 ms were applied thrice to determine the maximal isometric tetanic force with a 60-s interval between stimulations.4.Histopathological examinationHematoxylin-eosin(HE)staining was performed to observe the morphological changes and calculate the average cross-sectional area(CSA)of muscle cells.Immunohistochemical staining of fast and slow MyHC was performed to calculate the CSA and relative content of fast and slow muscle cells.Masson staining and Sirius red staining were performed to calculate the collagen volume fraction.Immunohistochemical staining was performed to calculate the content and ratio of typeⅠ and type Ⅲ collagen.5.Molecular examinationWestern blotting was performed to detect the relative protein content of age-relatedβ-galactosidase(GLB1),p16,p21,p53,TRB3,muscle atrophy markers atrogin land MuRF 1,type Ⅰ and type Ⅲ collagen,p62,LC3,p-JNK,JNK,p-ERK,ERK,p-p38,p38.Co-immunoprecipitation was performed to detect the interaction between TRB3 and p62,MEK1+MEK2,MEK3+MEK6,MEK4/MKK4,MKK7.Results1.Establishment of natural-aging animal modelWild type C57/BL6 mice were fed with normal diet to 3 months and 18 months of age,respectively,as the young group and the old group.Compared with the young mice,the old mice showed sparse bleak hair,and were dull,slow,and slightly heavier.The relative contents of the age-related markers GLB1,p53,p21,and p 16 were significantly increased in the old mice than in the young mice.These changes suggested that we had successfully established the natural-aging animal model.2.Exercise capacity was markedly decreased in old miceThe forelimb grip strength,the inverted hanging time,and the exhaustive running time were significantly lower in the old group than in the young group,indicating that the exercise capacity of old mice was significantly decreased.3.Skeletal muscle cells,particularly fast muscle cells,were overtly atrophied in old miceCompared with the young group,the old group had significantly decreased percentage of gastrocnemius weight in body weight and ratio of gastrocnemius weight to tibia length.HE staining showed that the old mice exhibited clear variations in the size and arrangement of muscle cells,and had significantly decreased CSA of muscle cells.Immunohistochemical staining for slow and fast MyHC showed that the CSA of fast and slow muscle cells in the old group was respectively decreased and increased compared with the corresponding CSA in the young group.The ratio of the number of slow muscle cells to the number of fast muscle cells was increased significantly in the old group.Besides,the expression of atrogin 1 and MuRF 1 was significantly higher in the WT old group than in the WT young group.4.Interstitial fibrosis was clearly evident in aging skeletal musclesCompared with the young mice,the old mice showed significantly increased collagen volume fraction,contents of collagen I and collagen III,and ratio of collagen I content to collagen III content.5.TRB3 expression was related to atrophy and fibrosis of aging skeletal musclesCompared with the young mice,the old mice had significantly increased protein content of TRB3 in the skeletal muscles.The CSA of skeletal muscle cells was negatively related to TRB3 expression,whereas the collagen volume fraction was positively related to TRB3 expression.6.TRB3 knockout alleviated skeletal muscle cell atrophy in old miceCompared with the WT old mice,the TRB3-/-old mice had non-significantly increased percentage of gastrocnemius weight in body weight,non-significantly increased ratio of gastrocnemius weight to tibia length,comparatively more uniform arrangement of muscle cells,significantly increased CSA of total and fast muscle cells,non-significantly increased ratio of the number of slow muscle cells to the number of fast muscle cells,significantly decreased expression of atrogin 1,and non-significant decreased expression of MuRF 1.7.TRB3 knockout reduced skeletal muscle interstitial fibrosis in old miceCompared with those in WT old group,the collagen volume fraction,the contents of collagen Ⅰ and collagen Ⅲ,and the ratio of collagen I content to collagen III content were decreased significantly in the TRB3-/-old group.8.TRB3 knockout enhanced the exercise capacity and the muscle function in old miceCompared with the WT old mice,the TRB3-/-old mice showed significantly increased forelimb grip strength,inverted hanging time,and EDL tetanic force,and a tendency of increased exhaustive running time and EDL twitch force.9.TRB3 knockout reduced skeletal muscle cell atrophy possibly by improving autophagyThe ratio of LC3-II to LC3-I and the content of p62 in skeletal muscles were significantly higher in the WT old group than in the WT young group,and were significantly lower in the TRB3-/-old group than in the WT old group.Co-immunoprecipitation experiments demonstrated the binding of TRB3 to p62 in skeletal muscles.10.TRB3 knockout reduced skeletal muscle fibrosis probably by affecting MAPK signaling pathwayRelative to the levels in the WT young group,the expression of TRB3 and the phosphorylation of JNK and ERK were significantly elevated in the WT old group,and the phosphorylation of p38 exhibited a decreasing tendency.Compared with those in the WT old group,TRB3 expression and JNK phosphorylation were decreased significantly,ERK phosphorylation was decreased non-significantly,and p38 phosphorylation was increased significantly in the TRB3-/-old group.Co-immunoprecipitation experiments demonstrated that TRB3 bound to MAPKKs,including MEK1/MEK2,MEK3/MEK6,and MEK4/MKK4 in skeletal muscles.However,no direct association were detected between TRB3 and MKK7Conclusions1.TRB3 was predominantly expressed in the skeletal muscles of old mice2.TRB3 knockout alleviated muscle cell atrophy,reduced interstitial fibrosis,and improved the exercise capacity of old mice.3.TRB3 knockout improved the obstructed autophagy flow,decreased the phosphorylation of JNK,increased the phosphorylation of p38,and thus had a protective effect on senile sarcopenia.BackgroundWith the acceleration of population aging,a series of demand for medical care services as well as economic burdens has been brought,which is a severe challenge facing the world.Sarcopenia is widespread in the elderly and has become a research hotspot in the field of geriatrics.Sarcopenia is an age-related syndrome characterized by progressive,generalized loss of muscle strength and muscle mass,which leads to many adverse events and is associated with poor prognosis of a variety of diseases However,the exact pathogenesis of sarcopenia remains to be further elucidated.Increased interstitial adipose in skeletal muscles is a major pathological change of sarcopenia.As special ectopic adipose tissue,the interstitial adipose has paracrine and autocrine functions.It changes the muscle microenvironment,interacts with muscle fibers,leads to muscle dysfunction,and is closely related to the decrease of exercise endurance,tension,strength and motor function.As is reported,age is an independent risk factor for the increase of intermuscular adipose.It increases by 9-70 grams per year,which is a key reason for the decrease of muscle function after aging.However,how the adipose increases in aging muscles is still unclear.Increased adipogenic differentiation of muscle-derived mesenchymal progenitors might be a key reason for increased adipose deposition in aging skeletal muscles.Multiple in-vivo and in-vitro studies confirmed that the mesenchymal progenitors are the major source of ectopic adipocytes in muscles.They are resident multipotent cells of mesenchymal origin,which can proliferate and differentiate abnormally under pathological conditions,resulting in increased adipose tissue and impaired myogenesis.Bone marrow-derived mesenchymal stem cells are of the same origin as muscle-derived mesenchymal progenitors.They are reported to have increased adipogenic capacity and decreased osteogenic capacity after aging,which was an important reason for osteoporosis and bone marrow lipid deposition in elderly people.Given their homology,senescent muscle-derived mesenchymal progenitors might behave similarly.However,whether their adipogenic capacity increases with aging and the mechanisms remain unclear.Pim 1 may act as a key molecule in regulating the adipogenic differentiation of mesenchymal progenitors.Microarray detection showed that Pim1 was expressed at high levels in aging muscles.Pim1 protein has serine/threonine kinase activity and is involved in regulating proliferation,apoptosis,differentiation,metabolism,and many other life processes.Pim1 was associated with fat content in cow muscles.It was highly expressed in both benign and malignant adipocyte tumors,but not in non-adipocyte tumors.Besides,Pim1 inhibitor inhibited the adipogenic differentiation of 3T3-L1 pre-adipocytes.All the above studies indicated that Pim1 might be a key target regulating the adipogenic differentiation of senescent mesenchymal progenitors.In this study,we established the natural-aging models of wild-type and Pim1-knockout mice and the replicative-senescent model of muscle-derived mesenchymal progenitors,and investigated whether and how Pim1 affected the adipogenic differentiation of muscle-derived mesenchymal progenitors and the adipose deposition in aging muscles.Objectives1.To investigate whether Pim1 intervention alleviates sarcopenia in aging mice;2.To investigate whether Pim1 intervention influences intermuscular adipose content in aging mice;3.To investigate the mechanisms by which Pim1 intervention affects intermuscular adipose content in aging mice;4.To investigate the mechanisms by which Pim1 intervention mediated changes in intermuscular adipose content influences sarcopenia in aging mice.Methods1.AnimalsFour-week-old male wild type(WT)and Pim1 knockout(Pim1-/-)mice were randomized into young and old groups.The young-and old-group mice were raised until they were 3 and 18 months old,respectively.2.Dual-energy x-ray absorptiometry body composition analysisAt the end of the experiment,the mice were weighed,anesthetized,and fixed onto a foam board.Fat and lean mass were measured by dual-energy x-ray absorptiometry equipment.3.Exercise capacityExercise capacity was evaluated at the end of the experiment as follows.Forelimb grip strength test:The mice were trained to grasp the horizontally-placed dynamometer,and were pulled gently horizontally backwards until they release the dynamometer.Each mouse was tested trice and the values were averaged.Hanging grid test:The mouse was placed in the center of a grid and then the grid was turned upside down with the mouse head declining first.Hanging time was recorded when the mouse fell off the grid.Each mouse was tested thrice with a>30-min interval between tests.The hanging time was averaged and the hanging impulse was calculated.Exhaustive running test:The mouse was placed on the treadmill track.The running started at 5 m/min with a slope angle of 0°,and then the speed and slope angle were increased by 5 m/min and 5° every 5 minutes until they reached 20 m/min and 14°,respectively.The exhaustive running time was recorded.4.Muscle functionAt the end of the experiment,the mouse was anesthetized,the extensor digitorum longus was exposed,and a surgical suture was tied to the distal tendon.Then the tendon was dissected and the muscle was separated up to the proximal attachment.The other end of the suture was tied to a force transducer at a low base tension.The muscle was connected to an isolated pulse stimulator via platinum wires.Twenty V/cm 5-ms square wave stimulation was administered and the base tension was gradually increased until the maximal wave occurred.With the tension state maintained,the muscle was stimulated trice with 20 V/cm 5-ms pulses with an interval of 5 s between stimulations,and the maximal isometric twitch forces were recorded and averaged.Then the muscle was stimulated trice with 5-ms pulses at 100 Hz for 300ms with a 60-s interval between stimulations,and the maximal isometric tetanic forces were recorded and averaged.Subsequently,the muscle was isolated and transected,and the maximal cross-sectional area was determined using a stereoscope and the Image Pro Plus.5.Tissue processingAfter weighing and anesthetizing,the blood was collected and the serum was separated and preserved at-80℃.Bilateral gastrocnemius muscles were collected and weighed.One muscle was stored at-80℃ after liquid nitrogen treatment.The other was fixed with 4%paraformaldehyde,dehydrated by gradient ethanol series,and embedded with paraffin to make 5-μm sections;or dehydrated by gradient sucrose series,and embedded with optimal cutting temperature compound to make 10-μm sections.6.Serological testsSerum was analyzed for free fatty acid,total cholesterol,high density lipoprotein cholesterol,low density lipoprotein cholesterol,and triglyceride using the blood chemistry analyzer,and was analyzed for adiponectin and leptin using the ELISA kits.7.Histopathological examinationHematoxylin-eosin staining was performed to observe the morphological changes Immunohistochemical staining of myosin,fast MyHC and slow MyHC was performed to calculate the CSA and relative content of fast and slow muscle cells.Immunohistochemical staining of laminin was performed to calculate the content of central nucleated cells.Oil red O staining was performed to detect the content of intermuscular adipose.Immunohistochemical staining of TNF-α and IL-6 was performed to evaluate the inflammatory status in skeletal muscles.8.Molecular detection of skeletal musclesWestern blotting was performed to detect the relative protein contents of senescence-associated β-galactosidase(GLB1),p16,p53,Pim1,adiponectin,muscle atrophy markers atrogin 1 and MuRF 1,early myogenesis markers MyoD and Myf5,late myogenesis marker MyoG,adipogenic transcription factors PPARγ,C/EBPβ,C/EBPδ and FABP4 in skeletal muscles.9.Separation and identification of PDGFRα+mesenchymal progenitorsPDGFRα+mesenchymal progenitors were isolated from skeletal muscles of 1-month-old mice using magnetic beads,and the purity was determined by immunofluorescent staining.10.Establishment of replicative-senescent model of PDGFRα+mesenchymal progenitorsPDGFRα+mesenchymal progenitor were passed once every week,and the medium were changed twice weekly.Cells at passage 6 and 22 were used as young and senescent cells,respectively.Senescence was evaluated by the following methods.Senescence-associated β-galactosidase activity assay was performed to detect the proportion of senescent cells.Western blotting was performed to detect the relative protein content of senescence-related markers.Real-time quantitative PCR was performed to determine the relative telomere length.11.Adipogenic induction of PDGFRα+mesenchymal progenitorsThe young and senescent cells were seeded at appropriate density and induced by adipogenic medium.The medium was changed every other day,and the adipogenic differentiation was evaluated in order to screen the optimal induction time.The young and senescent cells were cultured in adipogenic medium containing different concentrations of Pim1 inhibitor SGI1776,and the adipogenic differentiation was evaluated to screen the optimal concentration for inhibiting adipogenesis.12.Detection of adipogenesisOil red O staining was performed to observe the morphological changes.After extraction with isopropanol,OD510 was detected to evaluate lipid formation.Western blotting was performed to detect the relative protein content of adipocyte marker adiponectin.ELISA was performed to determine the content of adiponectin in the supernatant.13.The mechanisms by which Pim1 intervention affects the adipogenic differentiationThe young and senescent cells were cultured in normal adipogenic medium,adipogenic medium containing DMSO,or adipogenic medium containing 5-μM SGI1776.Western blotting was performed to detect the relative protein content of adipogenic transcription factors PPARγ,C/EBPβ,C/EBPδ and FABP4.14.The effect of Pim1 inhibition on the senescence of PDGFRα+mesenchymal progenitorsThe young and senescent cells were cultured in normal growth medium,growth medium containing DMSO,or growth medium containing 5-μM SGI1776.Senescence-associated β-galactosidase activity assay was performed to detect the proportion of senescent cells.Western blotting was performed to detect the relative protein content of senescence-related markers.15.The effects of Pim1-mediated adipogenic differentiation of PDGFRα+mesenchymal progenitors on in-vitro-cultured muscle cellsC2C12 cells were induced to differentiate into muscle cells.Young and senescent PDGFRα+ mesenchymal progenitors were firstly cultured with adipogenic medium containing DMSO or SGI1776 for 6 days,and then cultured with normal medium for 24 h.The supernatant was used as conditioned medium to culture the muscle cells Western blotting was used to detect the protein levels of muscle atrophy markers.Results1.Establishment of natural-aging model of wild-type and Pim1-knockout miceWT and Piml-/-mice were randomized into young and old groups and fed to 3 and 18 months of age,respectively.Compared with young mice,old mice showed sparse bleak hair,were dull and slow,and had significantly increased protein contents of GLB1,p53,and p16,indicating that we successfully established the natural-aging animal model.Compared with WT mice,Pim1-/-mice had significantly decreased protein level of Piml,indicating that we successfully established the Pim1 knockout mice model Compared with WT young mice,WT old mice had significantly increased protein level of Pim1 in the skeletal muscles.Compared with WTyoung mice,WT old mice showed significantly increased body weight and no obvious changes in serological parameters.Compared with WT old mice,Pim1-/-old mice exhibited significantly decreased body weight and significantly lower contents of leptin and free fatty acid in the serium.2.Pim1 knockout alleviated sarcopenia in aging miceWT old mice showed obviously decreased forelimb grip strength,hanging impulse,and exhaustive running time,which were significantly improved in Pim1-/-old mice,suggesting that Pim1 knockout enhanced the exercise capacity of aging mice.The tetanic force of extensor digitorum longus was significantly decreased in WT old mice compared with WT young mice,and was significantly improved in Pim1-/-old mice compared with WT old mice,indicating that Pim1 knockout enhanced the muscle function of aging mice.The lean mass and the percentage of gastrocnemius weight in body weight were significantly decreased in WT old mice compared with WT young mice,and were significantly increased in Pim1-/-old mice compared to WT old mice,indicating that Pim1 knockout improved the muscle mass of aging mice.The above results suggested that Pim1 knockout significantly alleviated sarcopenia in aging mice.3.Pim1 knockout alleviated intermuscular adipose content in aging miceWT old mice had significantly increased fat mass,intermuscular adipose content,and adiponectin protein content compared to WT young mice,whereas Pim1-/-old mice had obviously decreased fat mass,intermuscular adipose content,and adiponectin protein content compared to WT old mice,suggesting that Pim1 knockout alleviated intermuscular adipose content in aging mice.4.Pim1 positively regulates the adipogenic differentiation of PDGFRα+mesenchymal progenitors and promotes intermuscular adipogenesis4.1 Separation and identification of PDGFRα+ mesenchymal progenitors,and establishment of replicative-senescent cell modelPDGFRα+mesenchymal progenitors were separated using magnetic beads,and immunofluorescent staining of PDGFRa confirmed the high purity of them.Cells at passage 6 and passage 22 were used as young and old groups,respectively.Compared with the young group,the old group had significantly increased content of senescence-associated β-galactosidase positive cells,significantly increased protein contents of GLB1,p53,and p16,and significantly shortened relative telomere length,suggesting that we successfully established the replicative-senescent cell model.Compared with the young cells,the senescent cells had significantly increased protein level of Pim1.4.2 Senescent PDGFRα+mesenchymal progenitors had increased adipogenic potencyIn the young group,with the prolongation of induction time,the intracellular lipid droplets,the OD510,and the intracellular adiponectin protein content gradually increased in a time-dependent manner,and reached the peak on the 9th day.In the old group,with the prolongation of induction time,the intracellular lipid droplets,the OD510,and the intracellular adiponectin protein content also increased in a time-dependent way,and reached the peak on the 6th day.On the 3rd and 6th days,the senescent cells had obviously more intracellular lipid droplets and significantly larger OD510 compared to the young cells.The above results showed that the senescent cells were more sensitive to adipogenic stimulation and had increased adipogenic potency.Next,we examined the changes of adipogenic transcription factors.After adipogenic induction,the relative protein contents of PPARy,C/EBPβ,C/EBPδ,and FABP4 in young cells were increased in a time-dependent manner and reached peaks on day 9.In senescent cells,the relative contents were firstly increased with adipogenic differentiation,then reached peaks on day 6(except for PPARy),and finally decreased as the adipogenic differentiation ended.On day 6,the contents of C/EBPβ and C/EBPδwere significantly higher in senescent cells than in young cells.Considering the above results,we chose 6 days as the effective adipogenic induction time.4.3 Pim1 expression was up-regulated during the adipogenic differentiation of PDGFRα+mesenchymal progenitorsThe relative protein content of Pim1 was non-significantly increased in young cells over time.Senescent cells expressed high level of Pim1 protein on day 0.After adipogenic induction,the Pim1 content was firstly decreased possibly because the cells stopped growing,then increased with adipogenic differentiation,and finally decreased as the adipogenic differentiation ended.On day 6,the Pim1 protein content in senescent cells was significantly higher than that in young cells.4.4 Pim1 inhibitor significantly reduced the adipogenic differentiation of senescent PDGFRα+ mesenchymal progenitorsDifferent concentrations of Pim1 inhibitor SGI1776 were added to the adipogenic medium.The results showed that SGI1776 reduced the formation of intracellular lipids and reduced the OD510 in a concentration-dependent manner.Compared with the young solvent group,5-μm SGI1776 non-significantly decreased the OD510,significantly decreased the intracellular and supernatant adiponectin protein content in the young group.Compared with the old solvent group,5-μm SGI1776 significantly decreased the OD510,non-significantly decreased the intracellular adiponectin protein content,and significantly decreased the supernatant adiponectin content in the old group.The above results demonstrated that 5-μM SGI1776 significantly decreased the adipogenic differentiation of senescent PDGFRα+ mesenchymal progenitors.5.Pim1 promotes the adipogenic differentiation of senescent mesenchymal progenitors by activating the C/EBPδ adipogenic pathwaySenescent cells had significantly increased levels of C/EBPβ and C/EBPδ and significantly decreased levels of PPARγ and FABP4 compared to young cells;Pim1 inhibition drastically decreased the expressions of PPARy and FABP4 in young cells and the expressions of C/EBPβ and C/EBPδ in senescent cells.WT old mice exhibited increased content of C/EBPδ compared to WT young mice,whereas Pim1-/-old mice exhibited decreased content of C/EBPδ and FABP4 compared to WT old mice.The above results demonstrated that C/EBPδ might be the key molecule mediating the effect of Pim1 to regulate the adipogenic differentiation of senescent mesenchymal progenitors.In addition,Pim1 inhibition significantly decreased the expression of senescence-related markers in senescent PDGFRα+ mesenchymal progenitors,indicating that Pim1 inhibition might reduce the adipogenic differentiation of PDGFRα+ mesenchymal progenitor cells by making them younger.6.Decreased intermuscular adipose mediated by Pim1 knockout alleviated sarcopenia by inhibiting the atrophy of skeletal muscle fibers in aging miceHE staining showed marked variations in the size and arrangement of muscle fibers in WT old mice,but a comparatively more uniform arrangement in Pim1-/-old mice.Compared with WT young mice,WT old mice had significantly decreased total cross-sectional area(CSA)and CSA of fast muscle fibers,significantly increased ratio of slow to fast muscle fibers,and significantly increased protein contents of MuRF 1and atrogin 1.Compared with WT old mice,Pim1-/-old mice had obviously increased total CSA and CSA of fast muscle fibers,and obviously decreased protein contents of MuRF land atrogin 1,indicating that Pim1 knockout alleviated muscle fiber atrophy in aging mice.In-vitro experiments were conducted to verify whether the protective effects of Pim1 knockout on muscle cell atrophy was achieved by mediating the decrease of intermuscular adipose.The conditioned medium of senescent adipogenic PDGFRα+mesenchymal progenitors increased the protein levels of muscle atrophy markers in cultured muscle cells,whereas Pim1 inhibition alleviated the muscle cell atrophy caused by the conditioned medium of senescent adipogenic PDGFRα+mesenchymal progenitors.These results indicated that Pim1 knockout might play a protective role in ameliorating sarcopenia by reducing the generation of intermuscular adipose and reducing atrophy of muscle cells.Conclusions1.Pim1 knockout significantly alleviated sarcopenia in aging mice.2.Pim1 knockout alleviated sarcopenia in aging mice by decreasing the content of intramuscular adipose,and thereby reducing the atrophy of muscle fibers and the activation of satellite cells at the basal state.3.Senescent PDGFRα+ mesenchymal progenitors had increased adipogenic potency,which is an important reason for the increase of intermuscular adipose in aging skeletal muscles.4.Pim1 inhibition or knockout reduced the adipogenic differentiation of senescent PDGFRα+ mesenchymal progenitors by inhibiting the C/EBPδ pathway,thereby reducing the intermuscular adipose content in aging mice. |
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