The coronavirus-19 disease (COVID-19) pandemic reminds us of the importance of immune function, even in immunologically normal individuals. Multiple lifestyle factors are known to influence the immune function.The aim was to investigate the association between NK cell activity (NKA) and multiple factors including vitamin D, physical exercise, age, and gender.This was a cross-sectional association study using health check-up and NKA data of 2,095 subjects collected from 2016 to 2018 in a health check-up center in the Republic of Korea. NKA was measured using the interferon-γ (IFN-γ) stimulation method. The association of NKA with 25-(OH)-vitamin D (25(OH)D) and other factors was investigated by multiple logistic regression analysis.P = 0.035). In women, both low exercise  and medium to high exercise  decreased the risk compared to lack of physical exercise. Interestingly, in men and women older than 60 years, physical exercise significantly decreased the risk. Older-age was associated with increased risk of very low NKA in men, but not in women.The average age of subjects was 48.8 ± 11.6 years . Among 2,095 subjects, 1,427 had normal NKA (NKA ≥ 500 pg IFN-γ/mL), while 506 had low NKA (100 ≤ NKA < 500 pg/mL), and 162 subjects had very low NKA (NKA < 100 pg/mL). Compared to men with low 25(OH)D serum level (< 20 ng/mL), vitamin D replete men (30–39.9 ng/mL) had significantly lower risk of very low NKA (OR: 0.358; 95% CI: 0.138, 0.929; Physical exercise and vitamin D were associated with NKA in a gender- and age-dependent manner. Age was a major risk factor of very low NKA in men but not in women. The coronavirus-19 disease (COVID-19) pandemic caused by SARS-CoV-2 prompted us to keep our immune function healthy to protect the body from the pathogen. Physical exercise, nutrition and emotional status were frequently named as the main factors that can influence the immune function. Thus, lifestyle that includes regular physical exercise, healthy dietary and emotional habits were emphasized besides wearing masks and social distancing .in vitro, increased number of CD8+ T lymphocytes and so on were associated with increased mortality in the elderly , poor T-cell proliferation response to mitogens  elderly –9. Howev elderly , 11. Mos elderly . Therefo elderly , colorec elderly , and pro elderly , 16.dim NK cells, which mediate cytotoxicity, were depleted in ventilator-dependent patients, and CD56bright NK cells, that is immune-regulatory producing IFN-γ, were significantly depleted in all COVID-19 patients  who had undergone a comprehensive health check-up and NKA test at Chaum Life Center  were screened Figure 1Every individual undergoing health check-up was asked to fill out a questionnaire sent by mail 2 weeks before and to submit it on arrival. The questionnaire asked medical and social history and lifestyle habits including alcohol intake habits, smoking habits and physical exercise habits. Regarding physical exercise habits, it asked about exercise frequency  and exercise duration per workout session .For alcohol intake habits, individuals who did not drink at all or those who drank once a month or less frequently with a limit of 1–2 standard servings per session were defined as non-drinkers. Individuals who drank up to 3 times per week with a limit of 1–2 standard servings per session were defined as light drinkers. Individuals who drank more than light drinkers either in frequency or quantity were defined as heavy drinkers –29.For exercise habits, medium exercise was defined as exercise 2–4 times per week with 1–2-h duration per workout session or everyday exercise with less than 1-h duration per session –32. Low 2 (underweight), 18.5–27.5 kg/m2 , and ≥ 27.5 kg/m2 (obese), as suggested for Asian populations by the World Health Organization  was calculated by dividing body weight in kilograms by height in square meters. BMI was categorized with cut-offs of < 18.5 kg/mnization .® kit  containing a recombinant cytokine that specifically activates NK cells to release interferon-γ (IFN-γ)  . Accordifacturer –16 and dt-test or analysis of variance (ANOVA), and categorical variables were examined by Pearson’s Chi squared test. To identify the risk factors for or protective factors against low NKA, we compared the very low NKA group with the normal NKA group using multiple logistic regression analyses. Variables were eligible for entry into a multiple logistic regression model if they were significantly associated with P < 0.1 in univariable analyses or clinically important. Multiple logistic regression analyses were performed in gender, age, and exercise groups to find differential influences of relevant factors in each subgroup. Correlation was tested with Pearson’s correlation test. IBM SPSS statistics version 21  was used for all statistical analyses. Box plots were created using the R software (version 3.5.1).For univariable analyses, continuous variables were assessed using Student’s Among the initially screened 3,066 electronic medical records, 486 records were excluded due to multiple check-ups. Thereafter, 485 subjects were excluded according to the predefined exclusion criteria as already described above. Finally, 2,095 subjects were included for analyses Figure 1P = 0.047). In the very low NKA and low NKA group, the proportions of uncontrolled blood glucose level represented by Hb A1c ≥ 7.5 were higher than the normal NKA group . In the very low NKA and low NKA group, the proportions of vitamin D deficiency represented by serum level of 25(OH)D < 20 were higher than the normal NKA group though it could not reach statistical significance . The distributions of NKA value according to age and 25(OH)D are depicted in The mean (± SD) age of the study population was 48.8 ± 11.6 years, 52.9% were female, and the mean (± SD) NKA value was 1,243 ± 1,044 pg INF-γ/mL. Population characteristics according to NKA values are provided in P = 0.035). But this association was not observed in women (Compared to 25(OH)D < 20 ng/mL, the 25(OH)D 30–39.9 ng/mL significantly decreased the risk of very low NKA in men  and medium to high exercise  decreased the risk of very low NKA in women . The mean value of 25(OH)D level increased gradually from the no-exercise group (19.2 ± 9.92 ng/mL) to low exercise (22.4 ± 10.8 ng/mL), medium exercise (23.3 ± 10.0 ng/mL), and high exercise group (24.9 ± 10.5 ng/mL) (We checked the correlation between 25(OH)D (continuous variable) and exercise . 25(OH)D had a significant correlation with exercise (5 ng/mL) Figure 5P = 0.049).To investigate the independent association of vitamin D with NKA, we performed multiple logistic regression analyses in subgroups divided by exercise Table 3.P = 0.004) to age 61–70 years  and age ≥ 71 years   (In men with age ≤ 60 years (age 18–60 years), 25(OH)D level 30–39.9 ng/mL significantly decreased the risk of very low NKA  Table 4.P = 0.048) (P = 0.001) and medium to high exercise  decreased the risk significantly  Table 5.We investigated how age, gender, vitamin D, and physical exercise were associated with immune function determined by NKA. The association of each factor with NKA varied widely according to age and gender. Age was the determining factor for the very low NKA in men but not in women. Sufficient level of vitamin D reduced the risk for very low NK in men, and physical exercise reduced the risk in women. In men with age < 60 years, sufficient level of vitamin D reduced the risk for the very low NKA. In the subjects with age ≥ 60, physical exercise reduced the risk both in men and women. DM is well-known to diminish immune function, but neither DM nor hyperglycemia represented by Hb A1c showed significant association with NKA when adjusted for other factors. To the best of our knowledge, this is the first observation demonstrating age- and gender-specific association of vitamin D and physical exercise with NKA.In vitro, 65 ng/mL 25(OH)D has been found to be more effective than 30 ng/mL or 90 ng/mL in inducing human NK cell-mediated antibody-dependent cell cytotoxicity , poor T-cell proliferation response to mitogens in vitro, and increased number of CD8+ T lymphocytes representing effector-memory T cells) was associated with mortality  grant funded by the Korea government (MSIT) (No. 2020R1F1A107714812).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we elucidated the role of the CB2R in physiological brain aging. We used CB2R−/− mice of progressive ages in a behavioral test battery to assess social and spatial learning and memory. This was followed by detailed immunohistochemical analysis of microglial activity and morphology, and of the expression of pro-inflammatory cytokines in the hippocampus. CB2R deletion decreased social memory in young mice, but did not affect spatial memory. In fact, old CB2R−/− mice had a slightly improved social memory, whereas in WT mice we detected an age-related cognitive decline. On a cellular level, CB2R deletion increased lipofuscin accumulation in microglia, but not in neurons. CB2R−/− microglia showed an increase of activity markers Iba1 and CD68, and minor upregulation in tnfa and il6 expression and downregulation of ccl2 with age. This was accompanied by a change in morphology as CB2R−/− microglia had smaller somas and lower polarity, with increased branching, cell volume, and tree length. We present that CB2Rs are involved in cognition and age-induced microglial activity, but may also be important for microglial activation itself.Physiological brain aging is characterized by gradual, substantial changes in cognitive ability, accompanied by chronic activation of the neural immune system. This form of inflammation, termed inflammaging, in the central nervous system is primarily enacted through microglia, the resident immune cells. The endocannabinoid system, and particularly the cannabinoid receptor 2 (CB Inflammaging, low-grade age-dependent inflammation, has been named one of the seven pillars of aging ,2,3 and In the brain, inflammaging affects the activity of the resident innate immune cells—microglia. In young mice, microglia scan their surroundings to react to changes in the environment. Upon detection of neuronal damage or assault, they travel to the site of injury to phagocytose debris and to potentially induce a neuroinflammatory signaling cascade. However, this process is disturbed with aging. Aged microglia are less motile and have deficits in their phagocytic capacity, but show increased secretion of pro-inflammatory cytokines . This ag1R and CB2R); their ligands the endocannabinoids (ECs) 2-arachidonoylglycerol and anandamide, as well as EC-synthesizing and -degrading enzymes. Presynaptic CB1Rs are an integral part of a synaptic feedback mechanism [2Rs modulate immune cell functions and microglia activity. Under basal conditions, CB2R expression in the brain is low and not readily detectable with most conventional methods [2Rs on neurons [Many studies indicated that the endocannabinoid system (ECS) is an important regulator of microglial activity ,9,10 andechanism , whereas methods ,14,15,16 methods . Moreove neurons ,20,21,221R exhibit accelerated age-related cognitive decline, gliosis, and increased expression of inflammatory cytokines in the brain [1R binding to G-protein [9-THC, a CB1/CB2 agonist, resulted in recovery of their cognitive impairment to the levels of 2-month old mice [2Rs contribute to brain aging.Mice lacking CBhe brain ,24,25,26-protein ,28,29. Aold mice . The chaold mice . While t2R in physiological brain aging, focusing on cognition and inflammaging. We investigated cognitive performance of young, adult, and old CB2R−/− mice, and subsequently analyzed age-induced changes in microglial morphology and activity.In this study, we characterized the role of CB2R−/− mice, we used the partner recognition (PR) and Morris water maze (MWM). Anxiety-related behaviors were analyzed in the o-maze test (To investigate the age-related cognitive performance in CBaze test A. All exp < 0.0001 for each group). We detected no significant effects of genotype or age with regard to sociability. WT mice recognized their previous partner after 30 min separation and showed a preference for the novel partner in the 3-months and 12-months, but not in the 18-months group : 3-months WT mice p = 0.0468; 12-months WT mice p = 0.0035; 18-months old CB2R−/− mice p = 0.0472) . In contrast, the preference in CB2R−/− mice was higher than the chance level exclusively in the 18-month group. Consistently, after 1 h separation, we determined a preference for the novel partner in 3-month, but not in 12-months or 18-months old WT mice : 3-months WT mice p = 0.0021)  C. We als 0.0021) . We also2R−/− mice and WT controls showed a similar improvement during the acquisition phase and a similar performance during the reversal phase of the test  : WT mice: 3-months p = 0.0088; 12-months p = 0.0002; 18-months p = 0.0421; CB2R−/− mice: 3-months p = 0.0219; 12-months p = 0.0008; 18-months p = 0.0018)  D. Additi 0.0018) . Moreove 0.0018) . Taken t2,82 = 10.26; p = 0.0001; genotype effect: F1,82 = 4.427; p = 0.0384) . Post hoc analysis did not reveal any significant differences between genotypes within the same age group, but we noted a trend for increased distance travelled in the open compartment in CB2R−/− mice.In the o-maze test, we determined a significant age and genotype effect for the time spent in the open compartment (two-way ANOVA age effect: F 0.0384) A. Post h 0.0384) . Further2,81 = 4.152; p = 0.0192; genotype effect: F1,81 = 13.24; p = 0.0005). Additionally, we measured a genotype effect and a significant increase of head-dipping behavior in CB2R−/− mice in all age groups .Then, we assessed behaviors associated with anxiety and risk assessment B. An incWe next measured the accumulation of lipofuscin in hippocampal pyramidal neurons as age-related lipofuscin accumulation is associated with neuronal loss .2R mice   .We next analyzed the accumulation of lipofuscin in microglia from hippocampal stratum radiatum as age-related lipofuscin accumulation is also associated with microglial functional decline ,33.2R−/−  (2R−/− mice increased from 0.21% (3-months) to 3.35% (12-months) and reached 4.4% (18-months). The age-related increase of lipofuscin-accumulation was higher in CB2R−/− mice  which resulted in an interaction effect    A,B. In W 0.0006) B. Aligne 0.0001) C but was−/− mice .2R−/− mice to characterize microglial activity.Next, we analyzed Iba1 intensity and CD68 area fraction in the somas of hippocampal radial microglia from WT and CB2R−/− microglia with age    (2R−/− microglia from 18-months old mice (Iba1 intensity increased in both WT and CB 0.0001) A,B. This 0.0001) C. Intere 0.0305) B. This wold mice C.tnfa, il6, ccl2, arg1 and nos2 in the hippocampus as markers of inflammaging.We also analyzed the expression of the inflammatory mediators: tnfa    increased with age in the hippocampus. In WT mice, we observed a steady increase of tnfa expression. The expression of il6 increased from 3- to 12- months but then decreased from 12- to 18- months, back to the il6 expression levels at 3- months (Expression of = 0.002) A, il6 (t 0.0001) B and ccl- months B.tnfa expression was more prominent in CB2R−/− with significant increase between 3-months and 12-months and 3-months and 18-months .The expression of deletion D, wherea2R deletion did not majorly alter cytokine expression in the hippocampus, but subsided an age-related increase in the ccl2 expression.Thus, CB2R microglial activation in the context of inflammaging, we analyzed 3D microglial morphology of WT and CB2R−/− hippocampal microglia.To characterize the role of CB2R−/−  (2R−/− microglia as we detected a significant decrease in soma size in microglia from 18-months CB2R−/− old mice.The soma size of microglia significantly increased with age in both WT and CB 0.0001) B. Howeve2R−/− mice showed an altered morphology with significant differences in volume, ramification index, polarity index, tree length, and average branch length   C–G, whic2R deletion, in contrast to CB1R deletion, has little to no effect on age-related changes in cognitive or anxiety-related behaviors. Nevertheless, we detected subtle genotype effects on inflammaging and microglial function. CB2R−/− microglia exhibited an increased age-related lipofuscin accumulation and enhanced Iba1 and CD68 levels. Molecular changes were accompanied by altered microglial morphology, and moderately changed secretion of proinflammatory cytokines.We report that CBSeveral studies have shown that administration of THC to old animals was able to reverse many of the adverse consequences of aging on brain physiology and cognitive functions ,34,35. W2R on neurons prompted us to investigate if CB2R deletion also results in an accelerated aging phenotype with early cognitive impairment, similar to what has been observed in CB1R−/− mice. We found no evidence for an accelerated age-dependent memory decline in CB2R−/− mice. On the contrary, we observed that the changes of social memory were inversely correlated with age in CB2R−/− mice. While young CB2R−/− mice showed a slight decrease in social memory, old mice performed slightly better than age-matched controls. In agreement with the former, we have recently reported an impairment in social memory in CB2R−/− mice of both sexes aged between 4 and 6-months [2R−/− mice. The impairment in social memory in young mice did not arise due to altered anxiety-like behaviors. Contrary to previous reports, we measured an age-independent decrease in anxiety-like behavior in CB2R−/− mice [Recent reports confirming the presence of functional CB6-months . In cont−/− mice .2R−/− mice showed a decrease in hippocampus-dependent fear memory [2R−/− mice, as CB2R deletion decreased dendritic spine density in the hippocampus [In agreement with our findings, other studies done on young CBr memory ,37. Synapocampus ,38.2R age-related changes in the context of Alzheimer’s disease (AD), where modulating CB2R function impacted microglia activity, amyloid plaque load and cognitive abilities of AD-related model mice [2R on neurons or through microglia activity regulation remains unclear. Likewise, it is possible that either or both neuronal and microglial CB2Rs contributed to the social memory phenotype that we observed in our study. Nevertheless, our study strengthens an important role of CB2R in microglia activity in the context of inflammaging as we observed that the deletion of the CB2R increased accumulation of lipofuscin and CD68 levels in aged microglia.Previous studies have investigated CBdel mice ,40,41. W2R expression is increased in microglia and macrophages in many diseases and acute inflammatory states, but also during aging, which may be due to inflammaging [2R−/− mice had slightly increased cell surface, processes tree-length, number of branches, and ramification index, while their polarity index was decreased. These morphological changes were in line with a less-reactive microglial state and became less pronounced with aging. Microglial soma size increased with age, which is in line with previous studies [2R−/− microglia were smaller than those of WT microglia—especially in old mice—which could indicate reduced microglial activity. The aforementioned morphological changes were consistent with a less reactive state of microglia observed in young CB2R−/− mice. It is in agreement with previous findings showing a dampened immune response to a pro-inflammatory stimulus [2R signaling is required for an efficient microglia activation in young mice. Furthermore, a subtle decrease in an average branch length in addition to an increase in whole-cell Iba1 levels and CD68 levels, was detected in old mice. Age-related increases in Iba1 levels [2R−/− mice was accompanied by increased phagocytic activity. However, we did not observe corresponding changes in microglial morphology that would indicate a more reactive microglial state. Thus, it is possible that in CB2R−/− mice, the upregulation of Iba1 and CD68 did not result in a functional change of microglia.CBammaging ,42. Agedammaging ,43,44,45 studies . Howeverstimulus , thus in1 levels ,48 and e1 levels  were rep1R accelerated lipofuscin accumulation in the hippocampus [2R deletion on cognition could be a result of altered lipofuscin accumulation in either neurons or microglia. We detected an age-related increase of lipofuscin in both WT and CB2R−/− hippocampal microglia, as reported previously [2R−/− microglia showed enhanced lipofuscin accumulation, suggesting a deficit in lysosomal degradation. This idea is supported by a recent study that showed that CB2R activation promotes the autophagy flux in macrophages and that the autophagy-lysosome pathway was involved in CB2R-mediated HMGB1 (High mobility group box 1) degradation [2R in lysosomal pathways is still not well understood. Future studies should therefore include a more detailed analysis as to whether CB2R deletion impacts microglial phagocytosis and autophagy and whether these processes are also modulated by enhanced lipofuscin-accumulation.One of the key characteristics of aging is a disturbed proteostasis, which can be observed by an intracellular accumulation of potentially damaging protein aggregates . Among opocampus . Therefoeviously ,33,56. Hradation . Neverthtnfa, il6, ccl2, nos2, and arg1. Tnfa, ccl2, and il6 expression increased with age as reported previously [arg1 expression was not changed with age. In our study, il6 expression increased up to the age of 12 months and then decreased again at the age of 18 months. Since astrocytes also produce il6, we cannot exclude the possibility that astrocytes might dilute the direct effects of microglia. Interestingly, the age-induced increase of tnfa expression was even more pronounced in CB2R−/− mice when compared to WT mice. In contrast, ccl2 expression was significantly lower in CB2R−/− mice than in WT at the age of 18 months. We observed similar effects before in vitro and in an AD mouse [2R deletion alters inflammaging.We also analyzed the expression of inflammatory mediators eviously ,58, wherAD mouse , support2R mediated signaling in microglial response during aging was previously investigated only in the context of age-related neuroinflammatory diseases (including Alzheimer’s disease). CB2R activation decreased microglial activity [2R deletion [2R activation/inhibition represents acute effects, whereas CB2R deletion represents chronic effects, which might be highly variable, especially during long-term processes such as inflammaging. We have recently shown that CB2R is necessary for toll like receptor (TLR)-mediated microglial activation [2R−/− mice had distinct gene expression patterns, disturbed downstream signaling, and failed to show morphological signs of reactivity [2R activation is not only able to shift microglial activity from a pro- to an anti-inflammatory state but is also necessary to induce microglial activation in general. These recent data suggest that the role of CB2R on microglial activation is crucial and significantly more complex than previously thought and therefore needs to be investigated more thoroughly.CBactivity ,59,60. Sdeletion ,41. Howetivation . Stimulaactivity . The fin2R deletion has no effects on long-term spatial memory but has mild effects on short-term social memory during aging. Furthermore, we showed an age-dependent increase of lipofuscin in CB2R−/− microglia but not in CB2R−/− neurons. Enhanced lipofuscin accumulation in CB2R−/− hippocampal microglia was accompanied by increased Iba1 and CD68 levels. Microglial morphology was not majorly altered with age as aging exclusively increased microglia soma size but did not alter other investigated parameters. However, CB2R−/− microglia showed morphological differences independent of age with increased cell volume, ramification index, and process tree length, and decreased polarity and soma size. We conclude that CB2R plays a role in cognition and microglial regulation in an age-dependent manner. Furthermore, our data suggest that CB2R deletion contributes to microglial activity and might be crucial for microglial activation itself.Taken together, we report that CB2R−/− mice has been previously described [2R−/− mice were bred homozygous and backcrossed to the C57BL/6J line every six generations to minimize the risk of genetic drift.The generation of CBescribed . C57BL/6ad libitum water and food access, under 12 h light-dark cycle (lights on at 09:00 a.m.). Cages were monitored daily and bedding, water, and food were changed weekly. Experiments were carried out with male mice at the age of around 3, 12, and 18 months.All animals were housed in specific-pathogen-free conditions in the main animal facility of the University of Bonn. After weaning, mice were housed grouped in standard laboratory cages, with an automatic ventilation system, and Care of the animals and conduct of the experiments followed the guidelines of the European Communities Directive 86/609/EEC and the German Animal Protection Law regulating animal research and were approved by the Landesamt für Natur-, Umwelt-, und Verbraucherschutz (LANUV NRW), Germany (AZ 84-02.04.2017.A231).A week before the first behavioral test, mice were single-housed and transferred to a room with a reversed light-dark cycle (lights off at 9:00 a.m.). Tests were interspersed with 7-day intervals. Groups with 3, 12, and 18 month-old mice were tested independently and analyzed using Ethiovision XT 8.5 and 13 . The experimenter was blind to the genotype.Partner recognition (PR) paradigm was used to assess social memory. The test was performed in an open-field box (44 cm × 44 cm) containing a thin layer (about 1 cm) of sawdust. For three consecutive days, mice were allowed to explore the arena freely for 10 min, and habituate to the environment. On the test day, mice underwent two trials. In trial 1, mice were given 9 min to freely explore the arena containing an object  and a grid cage  with an unfamiliar C57BL6/J male partner mouse. The can and the cage were in opposite corners, each placed about 6–7 cm from the wall. Partner mice were approximately 10 weeks old. Interaction was noted when the mouse nose point was within 2 cm of the cage/object. The time spent on top of any of the objects was deducted from the interaction time. After trial 1, mice were returned to their home cages for 30 min  or 1 h . Sociabit-test against the chance level. Values above 50% indicated that the mouse spent more time interacting with a partner than with an object. In trial 2 the metal can was replaced by a grid cage with a novel mouse and the test mouse was given 3 min to freely explore and interact with both caged mice. Preference for the novel mouse was calculated as: preference (%) = Tn/(Tf + Tn) * 100, where Tf is the time spent with the familiar mouse and Tn is the time spent with a novel mouse. A preference for the new partner was interpreted as evidence for social memory. To detect learning in each group, we analyzed if the preference for the novel partner deviated statistically from the chance level with a one-sample t-test against a hypothetical mean (50%). Mice with sociability ≤55% were excluded from the analysis. If partner mice showed any signs of aggression, they were excluded from the analysis.The mean sociability value was tested with a one-sample The zero maze consisted of a circular runway with a diameter of 47 cm and width of 5.6 cm, elevated 30 cm above the ground. It was divided into four equally sized compartments, two of which were enclosed by 24 cm high walls. Mice were allowed to explore the maze for 5 min. Light intensity was around 200 lx. Head-dipping in the open compartment and stretched-attend postures were counted manually as described previously .The Morris water maze (MWM) was used (Morris 1981) to assess spatial learning and memory. In this paradigm, mice learn to locate a submerged and invisible platform in a round basin filled with turbid water, based on spatial cues. The experiment consisted of three phases: acquisition (days 1–6), probe trial (day 7), and reversal phase (days 8–10). During the acquisition phase the hidden platform remained in a fixed location and animals swam four times per day from different entry points. Inter-trial interval time was 1 h. The cut-off time for each swim was 90 s. If the mouse located the platform within the time limit, then it remained on it for an additional 5 s before being taken out of the maze. Otherwise, after the time limit passed, the mouse was guided to swim to the platform and remained there for an additional 20 s. The decrease of the time required to find the hidden platform indicated spatial learning. In the probe trial, the platform was removed and the time spent in the platform-associated quadrant was measured for 90 s. For the reversal phase, the platform was placed into the opposing quadrant, thus necessitating a re-learning of the position. Similar to the acquisition phase, mice swam four times daily and the latency to the platform was recorded. One mouse that stayed close to the wall at all times was excluded from the analysis.w/v formaldehyde for 3.5–4 h on ice. Afterwards, left hemispheres were incubated overnight in 10% sucrose, followed by an overnight incubation in 30% sucrose. The hemispheres were then frozen in dry ice-cooled isopentane and stored at −80 °C. The hippocampus was dissected from the right hemisphere and snap-frozen in liquid nitrogen.Mice were anesthetized and perfused transcardially with PBS. Brains were hemisected and one hemisphere was post-fixed in 4% ® protocol. Briefly, frozen tissue was homogenized in 1 mL or 800 μL TRIzol . Tissue homogenates were centrifuged and mixed with 160 μL chloroform. RNA was precipitated with 400 μL ice-cold isopropanol, washed twice with 75% ethanol, and the resulting pellet was dried. Subsequently, RNA was incubated with 2 μL DNase buffer, 10U DNase I and RNase free water in a total volume of 20 μL for 30 min at 30 °C, followed by a DNase inactivation at 75 °C for 5 min. RNA samples were stored at −80 °C.Total RNA was isolated from PBS-perfused, right hemispheric hippocampi (n = 6 hemispheric hippocampi per genotype per age group) using the TRIzolFor cDNA synthesis, 1080 ng RNA was incubated for 5 min at 65 °C and then reverse transcribed at 42 °C for 50 min. A total volume of 20 μL included 4 μL first-strand buffer (Invitrogen), 2 μL 0.1 mol/l DTT, 1 μL 10 mmol/l dNTPs, 0,5 μL oligo(dT) 20 primer (Invitrogen), and 200 U Super-Script II reverse transcriptase (Invitrogen).® Gene Expression system. A 30 ng quantity of cDNA was used per reaction. A standard program was applied as follows: step 1 (1×) 95 °C, 10 min; step 2 (40×) 95 °C, 15 s and 60 °C, 1 min. TaqMan primer : hprt (Mm03024075_m1), tnfa (Mm00443258 _m1), il6 (Mm00446190 _m1), ccl2 (Mm00441242_m1), arg1 (Mm00475988_m1), nos2 (Mm00440502_m1).Analysis by qPCR of cDNA samples was performed using a BioRad CXF384 Cycler and ThermoFisher TaqManw/v bovine serum albumin (BSA), 2% normal goat serum, and 0.5% Triton X-100 in PBS. Blocked sections were incubated with primary antibodies for 48 h at RT in the dark and, after several washing steps, incubated with secondary antibodies for 4 h at RT. Finally, slices were incubated with 0.1 μg/mL DAPI for 15 min and mounted on a slide using Fluoromount-G™ Mounting Medium. The following antibodies were used: Iba1 (AB_839504), CD68 (AB_322219), goat-anti-rabbit AF647 (AB_2535813), and goat-anti-rat AF488 (AB_2534074). High-resolution images were acquired with a confocal laser scanning microscope (Leica TCS SP8) using a 63x water-immersion objective lens (NA = 1.2). In each experiment, two z-stacks  were acquired per mouse of the strata radiatum and pyramidale in the CA1 hippocampal region. Lipofuscin accumulation was measured as autofluorescence (576–640 nm).Mouse brains were sectioned coronally at a thickness of 50 μm using a cryostat. Five dorsal hippocampal sections per mouse were stained as free-floating sections. Sections were post-fixed in 4% PFA for 2 h at room temperature (RT). After three washing steps with PBS, slides were blocked overnight at 4 °C in 10% Quantitative cellular parameters were determined using ImageJ . Two z-stacks with at least 5 microglia/stack were analyzed for each animal. Stacks from 6 mice/genotype were analyzed per age group.The CD68 content was determined within each microglial soma. Briefly, maximum intensity projections of the CD68 channel z-stacks were generated using the ‘z project’ command. Images were binarized with the ‘threshold’ command. Mean grey value threshold was kept constant among all groups. The threshold used was determined as an average individual threshold of all WT images. The area fraction of CD68 signal was measured within each microglial soma using the ‘measure’ command.Microglial morphology was quantified using a custom-written ImageJ toolbox designed to reconstruct and analyze microglial cells, similar to previous studies from Plescher et al. (2018) and Schmöle et al. (2018) ,60. The Neuronal accumulation of lipofuscin was measured in the stratum pyramidale of the hippocampal CA1 region from a binarized max z-projection (7 image planes) with a defined start. Number of lipofuscin particles of a size >0.5 µm was counted using the ‘particle analyzer’ plugin in ImageJ. Density was calculated as the number of lipofuscin particles divided by area of selection. Lipofuscin levels were measured in the soma of single stratum radiale microglia using binarized maximum z-stack projections as described above for the CD68 area fraction with at least 12 cells per mouse.The somas of microglia were manually delineated using the ‘polygon selection’ tool and saved as ‘regions of interest’ (ROI). Iba1 intensity and soma size were measured within each ROI with the ‘measure’ command. Soma size was measured in both Lipofuscin and CD68 area fraction experiments and both datasets were pooled together.www.graphpad.com. Figures were created in Adobe Illustrator (v 24.0.2). For presentation, representative images were post-processed in ImageJ (Fiji) to adjust brightness and contrast. All images within one experiment were adjusted the same way. Behavioral data were analyzed using Ethiovision XT 8.5 and 13 .Microsoft Excel (v 16.43) was used for data analysis followed by statistical analysis and data visualization in GraphPad Prism version 7.0.0 and 9.1.2 for Mac, GraphPad Software, San Diego, CA, USA, t-test. For single microglia analysis, an outlier test was performed with ROUT = 5% prior to the analysis, with the detected outliers excluded. For expression analysis, an outlier test was performed with ROUT = 10% prior to the analysis, with the detected outliers excluded. Datasets with more than 20 points were depicted using a violin plot to precisely visualize the distribution of the data, whereas datasets with fewer than 20 points were depicted as scatter plots. Statistical significance was stated when p-value < 0.05 at a 95% confidence interval. Detailed results of statistical analysis are presented in For datasets consisting of more than two groups with two independent variables , two-way analysis of variance (ANOVA) was used followed by Sidak’s multiple comparison test. For MWM repeated measurement (RM) ANOVA was used. For PR, the mean of the group was tested against a theoretical mean (50) with one-sample


Docking simulation results showed that 1e had a better binding affinity through a unique hydrophobic interaction with a PTPN9 hydrophobic groove. Moreover, 1e ameliorated palmitate-induced insulin resistance in C2C12 cells. This study showed that 1e increases glucose uptake and suppresses palmitate-induced insulin resistance in C2C12 myotubes via PTPN9 inhibition; thus, it is a promising therapeutic candidate for treating type 2 diabetes.Ginkgolic acid (C13:0) (GA), isolated from Ginkgo biloba, is a potential therapeutic agent for type 2 diabetes. A series of GA analogs were designed and synthesized for the evaluation of their structure–activity relationship with respect to their antidiabetic effects. Unlike GA, the synthetic analog  Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder characterized by hyperglycemia resulting from the combination of resistance to insulin action, inadequate insulin secretion, and excessive or inappropriate glucagon secretion ,2,3. LonProtein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that are involved in numerous cellular processes, such as growth, differentiation, metabolism, and immune response . More spGinkgo biloba (Ginkgo), also known as the maidenhair tree, is a deciduous tree, which has existed on Earth for approximately 200 million years and is considered to be a “living fossil” -2-deoxyglucose  was added to the cells, which were then incubated at 37 °C for 1 h. After incubation, the cells were washed with pre-cold PBS, and the fluorescence intensity was measured using a fluorescence microplate reader  (Ex/Em = 485/535 nm). For cell imaging, 3T3-L1 pre-adipocytes were seeded into µ-Slide 8 wells , and their differentiation was induced. Mature 3T3-L1 adipocytes and C2C12 myotubes were prepared as described earlier. Glucose uptake images were obtained at Ex/Em = 488/516 nm using a confocal microscope .Glucose uptake assays were performed following previously described methods with slight modifications . In briew/v) fatty acid-free BSA at 30 °C at a pH of 7.4 with stirring; this was followed by filtration (0.22 µm). Differentiated C2C12 cells were starved in serum-free DMEM for 3 h. Then, insulin resistance was induced by incubating the cells with 250 µM PA–BSA conjugate in DMEM for 16 h.The preparation of PA–BSA conjugates was carried out following a previously described method with slight modifications . In brieTM, Cytivalifescience, Seoul, Korea) supplemented with 10% FBS and 1% penicillin at a density of 1.5 × 104 cells/well. After 24 h of incubation, the cells were co-transfected using the Cignal PPAR Reporter  Reporter kit, CCS-3026L) and pCMV6-hFFAR4-GFP  or with SRE plasmids (pGL4.33 (luc2P/SRE/Hygro), Promega, Madison, WI, USA) and FFAR4  using a Lipofetamine 3000 device  according to the manufacturer’s instructions. After 6 h of incubation, the transfected CHO cells were treated with various concentrations of the compounds or the positive coFtablntrol. Control cells were treated with 0.1% DMSO. After incubation for another 24 h, luciferase activity was measured using the dual-luciferase assay system  according to the manufacturer’s instructions. All the individual in vitro assays to determine the average efficiency were performed twice and in triplicate for each treatment.This was performed following a previously described method . In briep < 0.05) was assessed using a two-tailed unpaired t-test for comparisons between two groups and a one-way ANOVA was used for multiple comparisons, followed by Tukey’s test. . Data are represented as the mean ± standard deviation (S.D.).To identify significant differences, data were analyzed using GraphPad Prism 7 . After testing the normality of distribution using a Shapiro–Wilk test at α = 0.05, statistical significance (


Most craniofacial manifestations of neurofibromatosis type 1 (NF1) are considered as a result of tumor compression. We sought to determine salivary changes, caries, and periodontal complications in NF1 patients without tumors in the oral cavity.Eleven NF1 patients without tumors in the oral cavity and 29 matched controls without NF1 were enrolled in this case–control study. Demographic information, medical history, and data of intraoral examinations, including the Decayed, Missing, and Filled Teeth (DMFT) scores and Russel’s periodontal index (PI), were recorded. The functional salivary analysis was performed for sialometry, salivary pH values, and amylase activity. Ingenuity Systems Pathway Analysis (IPA) was conducted to identify mutually activated pathways for NF1-associated oral complications.P = 0.04), gingivitis , and decreased salivary flow rates . Periodontal destruction, salivary changes, and dental caries in NF1 patients were age-dependent. Subgroup analyses based on age stratification suggested that salivary flow rates and salivary amylase activities were significantly low in NF1 patients aged over 20 years and that salivary pH values, PI and DMFT scores were significantly high among NF1- controls aged over 20. All oral complications were not significantly presented in NF1 patients aged below 20 years. IPA analyses suggested that cellular mechanisms underlying NF1-associated oral complications involved chronic inflammatory pathways and fibrosis signaling pathway.NF1 patients were associated with periodontitis (OR = 1.40, 95% CI = 1.06–1.73, NF1 patients without tumors in the oral cavity presented a comparatively high prevalence of age-dependent oral complications, including periodontal destruction and salivary gland dysfunction, which were associated with chronic inflammatory pathogenesis. Neurofibromatosis is a complex group of syndromes which occurs due to the inactivation of the various tumour suppressor genes precipitating various complications in the body , 2. ClasThe NF1 gene is ubiquitously expressed in all cells of the body, but the phenotypic expression differs from cell to cell and within a cell through different developmental stages of life . Hence, Due to its rarity, there has been a lack of studies reporting the oral complications of NF1, with findings of these works including an asymmetrical enlarged face with multiple cutaneous/disseminated and plexiform neurofibromas , 13, 14.In this case–control study, 11 NF1 patients of NF1+ status that met the diagnostic criteria for NF1 by the National Institutes of Health , 13, andDemographic information and medical history were collected. After conglomeration of data, clinical examination was performed using a mouth mirror and William’s periodontal probe. For the intraoral findings, the Decayed, Missing, and Filled Teeth (DMFT) index and Russel’s periodontal index (PI) values were recorded. Accordingly, the diagnoses of gingivitis and periodontitis –23 were To evaluate their salivary flow rates, sialometry using whole unstimulated saliva was performed in the morning between 10 to 11 AM. Patients were instructed not to brush or use alcohol/ non-alcohol-based mouthwashes, consume food or liquids 3 hours before the examination. The individuals were asked to rinse their mouths with water and after waiting 10 minutes, the saliva was collected in a sterile test tube for one minute , 25. AccTo identify the salivary pH values and salivary amylase activity (1/min), 1 ml of whole unstimulated saliva was collected in a sterile test tube and the pH value of saliva was measured using a digital pH meter . The entire solution was made to 50 ml by adding sterile water. To this solution, 5 drops of 1% starch solution (QUALI-TECH CHEM Starch Indicator 1% Solution 99% PURE) and 2 drops of iodine reagent  were added along the walls of the test tube. After mixing thoroughly, the colour change of the solution into the blue was set as the index timepoint, and from that point, the time taken for the solution to become transparent was recorded .t tests. P-values less than 0.05 were considered to be significant. Age distribution of sicca syndrome and periodontitis among NF1 patients and NF1- controls were demonstrated, and the chi-square test was used to test the significance. The odds ratios (ORs) were derived to estimate the risk of sicca syndrome and periodontitis between the NF1 patients and NF1- controls [The comparison of the NF1 patients and matched controls (NF1-) for each age subgroup was done by using controls , 27–29. P-value visualization to identify underlying mechanisms among peripheral blood from patients with NF1, periodontium tissue isolated from patients with periodontitis, and parotid gland tissue from patients with sicca syndrome, compared with healthy controls. The DE RNA-seq was derived by normalizing the RNA expression level of patient samples with that of healthy controls. RNA-seq data with values of − log10(P) larger than 1.3 were considered significant, and positive z-scores indicated up-regulation while negative scores indicated down-regulation. The canonical pathway analysis was conducted with Ingenuity Systems Pathway Analysis (IPA) software . Additionally, the network between NF1 and periodontitis or sicca syndrome/Sjogren’s syndrome was constructed through Path Explorer and Molecular Activation Prediction (MAP) within IPA, based on reported gene–gene interactions in Gene Expression Omnibus.Canonical Pathway Analysis was conducted by comparing differentially expressed (DE) ribonucleic acid (RNA)-sequencing (RNA-seq) data registered in Gene Expression Omnibus of the National Centre for Biotechnology Information. We compared RNA-sequencing data from whole-transcriptome expression profiling using z-score and Out of the 11 NF1 patients, 5 were females and 6 were males. In these 11 individuals, 4 aged below 21 years, 4 aged between 21 and 40, and 3 aged over 40 years. Controls were matched for age (+/− 3 years ), gender, residing region, socio-economic status, personal and deleterious habits, and medications, including calcium channel blockers and caffeine. The case-to-control ratio was 1-to-2.6 (Appendix Table P = 0.0002)  , and 6.53 for NF1 patients versus 4.8 for controls aged over 41 . In contrast, the PIs were not significantly different among NF1 patients below 20 years old  , and 22.67 for NF1 patients versus 7.71 for NF1- patients aged over 41 . In contrast, the DMFT scores were not significantly different among NF1 patients and NF1- participants below 20 years old    Fig. A were noP = 0.005), and 0.17 for NF1 patients versus 0.29 for controls aged over 41 . On the contrary, the flow rates were not significantly different among NF1 patients below 20 years old   for NF1 patients versus 0.38 (min−1) for NF1- controls aged between 20 and 40 , and 0.29 (min−1) for NF1 patients versus 0.36 (min−1) for NF1- controls aged over 41 . But the times were not significantly different among NF1 + individuals below 20 years old  , and 8.27 for NF1 patients versus 7.17 for controls aged over 41 . In contrast, the pH values were not significantly different among NF1 patients below 20 years old   values being 11.28 for NF1, 10.66 for periodontitis, and 3.30 for sicca syndrome, respectively.IPA analyses indicated that the possible mechanisms underlying the correlation between NF1 with cutaneous neurofibromas and oral complications including (1) periodontal destruction, and (2) salivary gland dysfunction, involved multiple chronic inflammatory pathways Fig. A. As forPath Explorer performed using IPA suggested that the downregulation of NF1 gene in NF1 patients may induce periodontitis via upregulation of TNF Superfamily Member 13b (TNFSF13B) while linking to sicca syndrome or Sjögren's syndrome with upregulated TNFSF13B, promyelocytic leukaemia (PML) and signal transducer and activator of transcription 1 (STAT1) Fig. . These rIn this case–control study, NF1 patients were associated with periodontal disease, decreased salivary flow rates, compromised amylase activities, and high prevalence of caries, all in an age-dependent manner. Significantly high PI scores and DMFT scores, low unstimulated salivary flow rates and amylase activities, and high salivary pH values, were observed among NF1 patients aged over 20 years old. For those aged below 20 years, the above-mentioned measurables were not significantly different between NF1 patients and NF1- controls. Moreover, the difference in the means was the highest for those aged over 40, followed by those aged between 20 and 40, finally those aged below 20. With RNA-seq data from peripheral blood in NF1 patients, gingival tissues from patients in periodontitis, and parotid glands in patients with sicca syndrome, IPA analysis predicted that several chronic inflammatory pathways were involved for the association. These pathways included the fibrosis pathway, which may initiate periodontitis and sicca-like symptoms through the upregulation of TNFSF13B, PML and STAT1.Although a case of gingival neurofibroma in the attached gingiva has been reported , 31, so . The basic environment of saliva can also increase the proteolytic activity of the organism, which promotes the deposition of calcium phosphate, thereby placing NF1 patients at great risk of dental caries and periodontal destruction as a feedback loop [Apart from the above-mentioned theory that low-grade chronic inflammation in NF1 patients may initiate or exacerbate periodontal disease , a previack loop .Other factors contributing to periodontitis and caries in NF1 patients include fused tooth, misregulation of amelogenesis leading to enamel hypoplasia, microdontia , low levSeveral studies proposed mechanisms that can explain salivary gland dysfunction in NF1 patients or models. For instance, animal studies suggested that the NF1 gene played an important role in the organogenesis of salivary glands . LikewisMoreover, mutations of the NF1 gene were reported to bring about complications that resulted from collagen loss or inability to produce collagen fibers, with which the underlying fibrosis pathways may account for pulmonary complications of NF1, such as interstitial lung disease and pre-capillary pulmonary hypertension –45, lackThe major strengths of this study included matching between NF1 patients and NF1- controls, with which the potential effect of confounding factors such as age, gender , region,In conclusion, this study demonstrated age-dependent periodontal destruction and functional salivary changes correlating with carious lesions in NF1 patients, which were evidenced by intraoral examinations and functional salivary tests. These oral complications, instead of resulting from direct compression by neurofibromas, may develop following systemically upregulated chronic inflammatory pathways.


Viral infections increase the risk of developing allergies in childhood, and disruption of mucosal homeostasis is presumed to be involved. However, no study has reported a role for viral infections in such disruption. In this study, we clarified the mechanism of immunoglobulin A (IgA) overproduction in viral infections. Autopsies were performed on 33 pediatric cases, IgA and interferon (IFN)β levels were measured, and histopathological and immunohistochemical examinations were conducted. Furthermore, we cultured human cells and measured IFNβ and IgA levels to examine the effect of viral infections on IgA production. Blood IgA levels in viral infections were higher than in bacterial infections. Moreover, IFNβ levels in most viral cases were below the detection limit. Cell culture revealed increased IgA in gastrointestinal lymph nodes, especially in Peyer’s patches, due to enhanced IFNβ after viral stimulation. Conversely, respiratory regional lymph nodes showed enhanced IgA with no marked change in IFNβ. Overproduction of IgA, identified as an aberration of the immune system and resulting from excessive viral infection-induced IFNβ was observed in the intestinal regional lymph nodes, particularly in Peyer’s patches. Further, increased IgA without elevated IFNβ in the respiratory system suggested the possibility of a different mechanism from the gastrointestinal system. It is known that aberrations of the immune system, such as allergies, occur in childhood ,2,3 and Generally, interstitial cells in gastrointestinal regional lymph nodes express interferon (IFN)β, a type I interferon, upon stimulation by the bacterial flora. IFNβ alters normal dendritic cells into mucosal dendritic cells; these mucosal dendritic cells surface express A Proliferation Inducing Ligand and B cell Activating Factor, which are B cell activating molecules, causing B cells to differentiate into plasma cells so as to constantly produce immunoglobulin A (IgA). Recently, such excessive IgA production or IgA deficiency has drawn attention as a cause of aberrations of the immune system, including the development of type I allergies ,10. As wThe present study aimed to investigate whether differences in location among various lymph nodes infected with viruses might affect IgA production. We focused on regional lymph nodes in the gastrointestinal and respiratory systems and performed blood biochemistry and histopathology on pediatric autopsy cases that had succumbed to viral or bacterial infections, with a view to the homeostatic IgA production mechanisms. Furthermore, we created viral infection models in cultured cells of various types of regional and non-regional lymph nodes and assessed IFNβ expression and subsequent IgA production in each lymph node.n = 6/11) in the viral infection group, 23.0% (n = 3/13) in the bacterial infection group, and 22.2% (n = 2/9) in the non-infection group.We compared the clinical reference values of blood total IgA in childhood by age and those in the autopsy cases. The proportion of cases that showed higher total IgA levels than the clinical reference value was 54.5% (p < 0.05) and non-infection group (16–78 mg/dL (median = 50 mg/dL), p < 0.05) ) than the bacterial infection group (11–130 mg/dL ((median = 28 mg/dL),  < 0.05) .p = 0.401) and non-infection group (26–1051 ng/mL (median = 62 ng/mL), p = 0.342); however, no statistically significant differences were noted ) than the bacterial infection group (3–1240 ng/mL (median = 78 ng/mL), re noted .p < 0.05) and non-infection group (16.0–78.0 mg/dL (median = 50.0 mg/dL), p < 0.05) , the viral infection group exhibited higher levels (25.0–149.9 mg/dL (median = 70 mg/dL)) than the bacterial infection group (11.0–130.0 mg/dL (median = 28.0 mg/dL),  < 0.05) .n = 9/11), 46% of the bacterial infection group (n = 6/13), and 77% of the non-infection group (n = 7/9)  or lower in 81.8% of the viral infection group (n = 7/9) .No statistically significant difference in blood IFNβ levels was noted among the viral infection group (<1.2 to 4.24 pg/mL (median = 1.2 pg/mL)), bacterial infection group (<1.2 to 7.30 pg/mL (median = 1.2 pg/mL)), and non-infection group (<1.2 to 2.20 pg/mL (median = 1.2 pg/mL)).We investigated the histopathological changes in pulmonary hilar lymph nodes and small intestinal Peyer’s patches in the non-infection, bacterial infection, and viral infection groups. When observing pulmonary hilar lymph nodes by cause of infection, we noted intensive congestion in the non-infection group, but observed only a few edematous changes . The alvHE staining of intestinal Peyer’s patches in the non-infection group was “mostly negative” with respect to both congestion and edema . IFNβ imHE staining of pulmonary hilar lymph nodes in the bacterial infection group showed “congested” and “edematous” findings . The alvHE staining of pulmonary hilar lymph nodes in the viral infection group revealed a “high level of congestion” and “edema” , and theDifferent poly(I:C) concentrations were added to each lymph node interstitial cell type. As a result, an apparent elevation of IFNβ expression  was noted with 10 ng/mL of poly(I:C) in intestinal Peyer’s patch  interstitial cells. IFNβ expression was elevated in a poly(I:C) concentration-dependent manner; the addition of 10,000 ng/mL of poly(I:C) resulted in levels of 15.18–22.06 pg/mL (average = 19.36 pg/mL).In mesenteric lymph node interstitial cells, the addition of 1000 ng/mL of poly(I:C) elevated IFNβ levels . The IFNβ level upon addition of 10,000 ng/mL of poly(I:C) was 7.26–7.56 pg/mL (average = 7.38 pg/mL). Likewise, in the fossa axillaris lymph node -origin interstitial cells used as a control, the addition of 1000 ng/mL of poly(I:C) resulted in elevated IFNβ expression ; furthermore, the IFNβ level upon addition of 10,000 ng/mL was 5.94–6.33 pg/mL (average = 6.14 pg/mL). The rate of IFNβ elevation was high in intestinal Peyer’s patch  interstitial cells and low in fossa axillaris lymph node-origin interstitial cells .Pulmonary hilar node-origin interstitial cells treated with 100 ng/mL poly (I:C) exhibited IFNβ expression levels of 1.27–1.33 pg/mL (average = 1.29 pg/mL). This level was approximately twice the IFNβ level in the absence of poly(I:C), 0.62–0.68 pg/mL (average = 0.65 pg/mL). Furthermore, IFNβ expression was elevated in a poly(I:C) concentration-dependent manner; the addition of 10,000 ng/mL of poly(I:C) resulted in IFNβ levels of 6.27–6.41 pg/mL (average 6.34 pg/mL). Poly(I:C) treatment of the inguinal lymph node -origin interstitial cells, used as a control, exhibited similar results to the pulmonary hilar node -origin interstitial cells. The IFNβ level upon addition of 100 ng/mL of poly(I:C) was 0.95–0.98 pg/mL (average = 0.97 pg/mL), which was approximately twice the IFNβ level in the absence of poly(I:C) . IFNβ expression was elevated in a poly(I:C) concentration-dependent manner; the IFNβ expression level upon addition of 10,000 ng/mL of poly(I:C) was 5.72–5.86 pg/mL (average = 5.79 pg/mL) . HoweverIgA production levels were measured after the addition of different IFNβ concentrations to lymphocytes from various types of lymph nodes. The addition of 50 ng/mL of IFNβ resulted in apparent increases in IgA production  in intestinal Peyer’s patch  lymphocytes. Moreover, IgA production was increased in an IFNβ concentration-dependent manner; the addition of 10,000 ng/mL resulted in 146.6–153.3 ng/mL (average = 150.5 ng/mL). Similarly, IgA production in gastrointestinal regional lymph node lymphocytes also increased depending on the concentration of IFNβ. The addition of 50 ng/mL of IFNβ increased IgA production , and the addition of 10,000 ng/mL IFNβ resulted in peak IgA levels, 35.7–38.3 ng/mL (average 37.2 ng/mL) . ConversLymphocytes of pulmonary hilar lymph nodes, which are respiratory regional lymph nodes, produced 10.9–12.1 ng/mL (average = 11.5 ng/mL) of IgA upon addition of 50 ng/mL of IFNβ. This level was approximately 60 times the IgA level in the absence of poly(I:C), 0.2–0.3 ng/mL (average = 0.2 ng/mL). IgA production increased depending on the concentration of IFNβ, reaching 33.7–35.4 ng/mL (average = 34.4 ng/mL) upon addition of 10,000 ng/mL of IFNβ. In comparison, IgA levels in the inguinal lymph node  lymphocytes, used as a control, after the addition of IFNβ increased depending on the concentration of IFNβ, similar to the results of respiratory regional lymph node lymphocytes; the IgA level upon addition of 50 ng/mL of IFNβ was 3.1–3.3 ng/mL (average = 3.2 ng/mL), which represented approximately 33 times the IgA level in the absence of IFNβ . The IgA level reached 7.8–8.1 ng/mL (average = 7.9 ng/mL) upon addition of 10,000 ng/mL of IFNβ. However, the rate of increase was lower than that for pulmonary hilar lymph node  lymphocytes .As a result of the addition of IFNβ to lymphocytes, IFNβ concentration-dependent blast formation was noted in intestinal Peyer’s patch lymphocytes and pulmonary hilar lymph node lymphocytes .The present study aimed to investigate whether differences in location between various lymph nodes infected with viruses might affect IgA production. Focusing on gastrointestinal and respiratory regional lymph nodes, we explored the behavior of IFNβ and IgA associated with viral infections in various lymph nodes. The results suggested that in gastrointestinal lymph nodes, including intestinal Peyer’s patches, IgA production increased via excessive IFNβ expression resulting from viral infection, thereby disrupting the homeostatic environment of lymph nodes to play a role in immunological aberrations such as allergies.In autopsy cases where death was attributed to respiratory infection, viral infections exhibited higher blood total IgA and sIgA levels than in cases with bacterial infections. Given the fact that the majority of blood IgA is derived from bone marrow , the resMeanwhile, there was marked congestion and edema in the alveolar interstitia in the viral infection group. Strong IFNβ immunostaining of the intestinal lymph nodes was observed, whereas the pulmonary hilar lymph nodes showed weak staining. IgA immunostaining indicated that both the pulmonary hilar and intestinal lymph nodes were strongly positive. Furthermore, blood IFNβ levels in the viral infection group of autopsy cases did not increase compared with those in the non-infection group. This is probably because the increase in IFNβ levels in the lymph nodes was not reflected in the blood IFNβ levels, demonstrating the fact that IFNβ expression associated with IgA production occurs in lymph nodes . From thThe cell culture experiments using intestinal and respiratory lymph node lymphocytes and lymph node interstitial cells revealed that in intestinal Peyer’s patches , poly(I:C) stimulation caused IFNβ expression in the interstitial cells and that IgA production from lymph node lymphocytes increased in proportion to the amount of expressed IFNβ.Poly(I:C) stimulation of respiratory lymph node interstitial cells produced low IFNβ levels that were virtually identical to that observed for inguinal lymph node  interstitial cells. However, IFNβ stimulation of respiratory regional lymph node lymphocytes led to high levels of IgA secretion compared with that observed for inguinal lymph node lymphocytes, and the levels rapidly increased at 10 ng/mL of IFNβ in a concentration-dependent manner. That is, marked changes in IFNβ levels were not observed in respiratory regional lymph nodes with respect to viral infection stimulation, only IgA production resulting from IFNβ addition was increased in the respiratory regional lymph nodes. However, the number of intestinal infections  in actual pediatric autopsy cases is extremely low; thus, it was not possible to investigate intestinal infections in the present study. Therefore, it was not possible to elucidate whether increases in IgA secretion without the involvement of IFNβ, as noted in the pulmonary hilar lymph nodes in the present study, might be observed in all viral infections.Blast formation was observed in the cell culture experiment. Mature lymphocytes do not divide and proliferate, but when they encounter specific antigens, they adopt a pre-mature, juvenile cell format and start proliferating. This is called blast formation. Mature lymphocytes are classified as small lymphocytes, and juvenile lymphocytes are classified as large lymphocytes. Thus, the change in size observed in this experiment can be considered blast formation, an increase in the number of juvenile lymphocytes.The results of this study suggested that activation of the IFNβ signaling pathway, as mentioned above, might selectively occur in intestinal regional lymph nodes, rather than occurring simultaneously in all lymph nodes. The reasons why intestinal Peyer’s patches, which are intestinal lymph nodes, selectively respond to IFNβ and IgA activities remain to be fully elucidated. However, one hypothesis assumes that the immune system of Peyer’s patches, which have the structural peculiarity of not being covered by a membrane (unlike other lymph nodes) and are aggregations of lymph nodules having germinal centers where antibodies are actively produced , is so wThe present study revealed that enhanced IgA production, identified as an aberration of the immune system, in intestinal regional lymph nodes resulted from overexpression of IFNβ following viral infection stimulation, and IgA might be produced in the respiratory system via a different mechanism. The limitations of this study include the use of autopsy samples and the evaluation of monocultures, which does not allow understanding of the cell-to-cell relationship.A total of 33 pediatric autopsy cases  aged 0–12 years  was investigated. The case details are provided in Total blood IgA level was measured as follows: anti-human serum IgA was bound to 0.4 mL of a serum sample; the amount of IgA contained in the resulting complex was then measured in terms of turbidity at 694 nm using a turbidimetric immunoassay; and IgA levels in the serum sample were quantified. The reagent N-Assay TIA IgA-SH Nittobo  and an automatic analyzer JCA-BM8000 series  were used ,29,30. TThe sIgA enzyme-linked immunosorbent assay (ELISA) kit  ,32,33 waBlood IFNβ levels were measured using an IFNβ ELISA kit  ,35,36. TParaffin sections (4 μm) of the pulmonary hilar lymph nodes and the small intestine (duodenum or jejunum) including intestinal Peyer’s patches of the 33 autopsy cases were prepared and subjected to HE staining to investigate histopathological changes in the viral infection, bacterial infection, and non-infection groups.Furthermore, immunostaining was performed on the paraffin-embedded sections from the 33 autopsy cases through color development of 3,3′-diaminobenzidine using an anti-human IgA rabbit polyclonal antibody  and anti-human IFNβ rabbit polyclonal antibody  [In the experiments using cultured cells, Peyer’s patch interstitial cells and intestinal tract membrane lymph node-origin interstitial cells were used as gastrointestinal regional lymph node interstitial cells, and fossa axillaris lymph node -origin interstitial cells were used as controls.Intestinal Peyer’s patch lymphocytes and mesenteric lymph node lymphocytes were used as gastrointestinal regional lymph node lymphocytes, and fossa axillaris lymph node  lymphocytes were used as controls.Pulmonary hilar node-origin interstitial cells were used as respiratory regional lymph node interstitial cells. Inguinal lymph node -origin interstitial cells were used as controls. Pulmonary hilar lymph node lymphocytes were used as respiratory regional lymph node lymphocytes. Inguinal lymph nodes  were used as controls.2 concentration of 5%.Cells used in the aforementioned experiments were provided from the Laboratory of Clinical Regenerative Medicine, Faculty of Medicine, and University of Tsukuba. The cells provided were originally lymphocytes and interstitial cells separated from various lymph nodes by Lymphocyte Separation Medium 1077 . Therefore, they were not primary lymphocytes, but a cell line that was established by passaging those lymphocytes. Human lymph node interstitial cells and lymphocytes were cultured in 60 mm dishes containing 6 mL of Roswell Park Memorial Institute 1640 supplemented with human albumin for 72 h at 37 °C with a COPeyer’s patch interstitial cells and intestinal tract membrane lymph node-origin interstitial cells were used as interstitial cells of gastrointestinal regional lymph nodes, and fossa axillaris lymph node-origin interstitial cells were used as controls. Pulmonary hilar node-origin interstitial cells were used as interstitial cells of respiratory regional lymph nodes, and inguinal lymph node-origin interstitial cells were used as controls.Poly(I:C), which is a synthetic analogue of double-stranded ribonucleic acid (RNA) mimicking viral RNA and designed to simulate viral infections , was addIntestinal Peyer’s patch lymphocytes and mesenteric lymph node lymphocytes were used as gastrointestinal regional lymph node lymphocytes, and fossa axillaris lymph node lymphocytes were used as controls. Further, pulmonary hilar lymph node lymphocytes were used as respiratory lymph node lymphocytes, and inguinal lymph node lymphocytes were used as controls. IFNβ was added to each lymphocyte type at a concentration of 0, 10, 50, 100, 1000, or 10,000 ng/mL during lymphocyte culture. IgA levels were measured by ELISA after 24 h.IFNβ and IgA in the different cultured cell types were quantified using an ELISA kit  ,35,36 anThe nonparametric Mann–Whitney U test was used for comparisons between two groups. The Games–Howell test was used for comparisons between multiple factor groups. Analyses were performed using Microsoft Excel and IBM SPSS statistic viewer 24 ,42.The enhancement of IgA production, identified as an aberration of the immune system, in the gastrointestinal system is associated with increases in IFNβ expression triggered by viral infections. Moreover, IgA production in the respiratory system might be induced by a different mechanism.


Rosuvastatin (RST) is primarily used to treat high cholesterol levels. As it has potentially harmful but not well-documented effects on embryos, RST is contraindicated during pregnancy. To demonstrate whether RST could induce molecular epigenetic events in the brains of newborn rats, pregnant mothers were treated daily with oral RST from the 11th day of pregnancy for 10 days (or until delivery). On postnatal day 1, the brains of the control and RST-treated rats were removed for Western blot or immunohistochemical analyses. Several antibodies that recognize different methylation sites for H2A, H2B, H3, and H4 histones were quantified. Analyses of cell-type-specific markers in the newborn brains demonstrated that prenatal RST administration did not affect the composition and cell type ratios as compared to the controls. Prenatal RST administration did, however, induce a general, nonsignificant increase in H2AK118me1, H2BK5me1, H3, H3K9me3, H3K27me3, H3K36me2, H4, H4K20me2, and H4K20me3 levels, compared to the controls. Moreover, significant changes were detected in the number of H3K4me1 and H3K4me3 sites , which are generally recognized as transcriptional activators. Fluorescent/confocal immunohistochemistry for cell-type-specific markers and histone methylation marks on tissue sections indicated that most of the increase at these sites belonged to neuronal cell nuclei. Thus, prenatal RST treatment induces epigenetic changes that could affect neuronal differentiation and development. Statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors) are a class of lipid-lowering agents used in the treatment of high cholesterol levels . Apart fBesides their beneficial properties, statins also have numerous adverse effects . As a reRST, one of the highest-selling prescription drugs on the market , exhibits the greatest inhibitory effect on cholesterol biosynthesis , and amoHistone modification is one of the main mechanisms of epigenetic modification regulating gene expression. This modification requires several different histone-modifying enzymes, including “writers”, which attach modifications to histone tails, “erasers”, which remove modifications, and “readers”, which recognize these modifications ,29,30,31As recent studies have emphasized the importance of maternal effects on chromatin structure and the interrelationship between the genome, epigenome, and environment ,36, our When investigating epigenetic events in the newborn brain after the administration of RST, we assumed that liver pâté, the vehicle used to deliver RST, would not elicit changes in histone methylation patterns. To reveal such possible effects of the liver pâté, we assayed H2AK118me1, H2BK5me1, H3, H3K4me1, H3K4me3, H3K9me3, H3K27me3, H3K36me2, H4, H4K20me2, and H4K20me3 levels using Western blots from absolute control and vehicle-treated control newborn rat brain samples. Our data showed that liver pâté, as a nutritional supplement, had no significant effect on the methylation patterns of these sites . As all RST exposure in utero did not cause structural abnormalities in the newborn brain at the level of light microscopy as evidenced by H&E staining . Double Several antibodies that recognize different methylation sites for the H2A, H2B, H3, and H4 histones were used in the Western blotting experiments . We founCell-specific markers were used to localize the increases in H3K4me1- and -me3-immunopositivity in the cerebral tissue sections. Most of the immunoreactivity was localized to NeuN-positive neuronal cell nuclei, which constitute the vast majority of the parenchyma, and typically displayed the large nuclei of neuronal morphology . InteresAlthough the beneficial effects of statins are well documented , there aThe nervous system comprises several different cell types that are defined by morphology, function, anatomical location, and specific patterns of gene expression. The establishment of these complex and highly regulated cell fates requires the spatial and temporal coordination of gene transcription in the proper order, number, and location. In the developing brain, neurons are generated first, followed by the supporting glia. Lineage studies indicate that the developing brain contains multipotent progenitor cells capable of generating both neurons and glia, and that cell fate restriction may be a consequence of a series of gene expression events and extracellular signals acting on multipotent progenitors . On postnatal day 1, the newborn rat brain is a tissue populated mostly by neurons that are complemented gradually with non-neuronal cells. Bandeira et al.  reportedMammalian embryonic development and subsequent fetal development involve precise molecular interactions between intrinsic factors such as genetics and epigenetics, and extrinsic maternal factors, such as environmental perturbations, drugs, or even maternal nutrition, as nutritional components that could influence the epigenetic landscape in the fetus and thus developmental processes . EuchromThe dysregulation of chromatin decreases viability and normal cell functions and leads to various neurodevelopmental and psychiatric diseases . H3K4 meIn this study, we focused on methylations of the Lys (K) residues of the four core histones. These epigenetic modifications, particularly in the case of the histone H3, are involved in a wide range of biological processes, including the activation and repression of transcription; nevertheless, one methyl mark by itself might still have a limited biological message. As mentioned above, H3K4me is a mark that, on a genome-wide scale, is associated broadly with transcriptional activation and the epigenetic tagging of promoters and enhancers . Hence, In this study, we provided evidence that, in newborn rat brains, prenatal exposure to RST alters the methylation landscape in general, and the H3K4me1 and H3K4me3 histone modifications in particular. We observed that RST significantly elevated the levels of H3K4me1 and H3K4me3 compared to the controls. Furthermore, we did not find any histological structural alteration in newborn brains that could be related to RST treatment. It remains to be determined in future studies whether the changes observed in the H3K4 methylation patterns are: (1) linked to specific loci or are genome-wide, (2) reflect an adaptive or maladaptive response to RST, or (3) are the outcomes of secondary or tertiary processes in response to RST treatment. The identification of the genomic sequences involved in the control of the embryonic development of offspring whose mothers had been treated with RST remains a formidable challenge. Interestingly, statins, in general, did not directly inhibit the activity of the major epigenetic modifying enzymes, such as, “writers” or “readers” . Since 5Pregnant Sprague–Dawley rats (190–210 g) were kept under standard housing conditions and fed ad libitum with regular laboratory chow. Pregnant rats were divided into three groups: in addition to absolute controls , the vehicle-treated control animals received a small amount (650 mg) of liver pâté in pellet form once a day, whereas treated rats were given daily oral doses of RST (0.25 mg/kg body weight) mixed into pellets of liver pâté. Both groups received this liver pâté supplement (with or without RST) for 10 days, beginning on the 11th day of pregnancy (or until delivery). Feeding with the liver pâté was done individually, using forceps. Five breeding runs (4–6 pregnant rats each) provided the litters (6–12 pups from each mother) from which the independent experiments were performed. On postnatal day 1, the cerebral hemispheres of the absolute control, vehicle-treated control, and RST-treated rats were removed and either homogenized for Western blot analysis or embedded in paraffin for hematoxylin and eosin (H&E) staining and fluorescent immunohistochemistry/confocal microscopy.The antibodies used in this study are listed in Rat brains were dissected quickly, fixed in 0.05 M phosphate-buffered saline (PBS) containing 4% formaldehyde, and then embedded in paraffin for H&E staining and immunohistochemistry. Paraffin-embedded sections were cut (6 µm thickness) on a microtome , mounted on glass slides coated with (3-aminopropyl)triethoxysilane , and subsequently used for H&E staining and immunohistochemistry.The paraffin-embedded tissue sections were deparaffinized, rehydrated, and placed in a jar filled with 0.01 M citrate buffer (pH 6.0) containing 0.05% Tween-20, and then heated at 95 °C for 20 min. The sections were washed 3 × 10 min in 0.05 M PBS containing 0.05% Tween-20 and blocked in a 0.05 M PBS solution containing 0.05% Tween-20 and 5% normal goat serum (NGS) for 1 h at room temperature (RT). The sections were then incubated with primary antibodies in a 0.05 M PBS solution containing 0.05% Tween 20 and 5% NGS overnight at 4 °C. After washing (4 × 10 min in 0.05 M PBS containing 0.05% Tween-20), the primary antibodies were labeled with either Alexa 488- or Alexa 568–conjugated secondary antibodies  in a blocking solution for 3 h at RT. After 4 × 10 min washes in 0.05 M PBS containing 0.05% Tween-20, the cell nuclei were stained using a 2-[4-(aminoiminomethyl)phenyl]-1H-indole-6-carboximidamide hydrochloride (DAPI) solution . Digital images were captured on a Leica DMLB epifluorescence microscope using a Leica DFC7000 T CCD camera  and the LAS X Application Suite X (Leica).Z-axis, with a distance of 0.5 μm between consecutive optical slices using the following microscope configuration: objective lens, UPLSAPO 60×; numerical aperture, 1.35; sampling speed, 4 μs/pixel; optical zoom, 2×; and scanning mode, sequential unidirectional. The excitation wavelengths were as follows: 405 nm (DAPI), 488 nm (Alexa Fluor 488), and 543 nm (Alexa Fluor 568). Z-stack images were prepared using 10–12 consecutive optical slices.Select immuno-labeled sections were also examined with a confocal laser scanning microscope . Images (512 × 512 pixels) were captured along the g for 10 min. The supernatant was aliquoted, and the pellet was rehomogenized in 50 mM Tris-HCl (pH 7.5 at 4 °C) containing 150 mM NaCl, 0.1% Nonidet-P40, 0.1% cholic acid, 2 μg/mL leupeptin, 1 μg/mL pepstatin, 2 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA. The samples were incubated on ice for 30 min and then aliquoted. The protein concentrations of these suspensions and the abovementioned supernatants were determined by the Lowry method [Brains from newborn rats were dissected, homogenized in 50 mM Tris-HCl (pH 7.5 at 4 °C) containing 150 mM NaCl, 2 μg/mL leupeptin, 1 μg/mL pepstatin, 2 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA, and centrifuged at 14,000× y method . For Weshttps://imagej.net/Downloads; accessed on 10 July 2013) [Grayscale digital images of the immunoblots were acquired by scanning the autoradiographic films with a desktop scanner . The images were scanned and processed at identical settings to allow comparisons of the Western blots obtained from different samples. The bands were analyzed by densitometry via ImageJ  by W. Rasband, available at ly 2013) . The immp-value of < 0.05 was considered significant.All statistical comparisons were made using SigmaPlot software , and data were analyzed with one-way analysis of variance (ANOVA) or the Mann–Whitney rank–sum test. For the Western blots, values were presented as means ± standard errors of means (SEMs) from at least five immunoblots, each representing an independent newborn. A


Normal Sprague-Dawley rats were chosen as nondiabetic control group. Urinary albumin/creatinine ratio (ACR), biochemical parameters, renal histopathology and podocyte apoptosis, and morphological changes were evaluated. Expressions of mitochondrial dynamics-related and autophagy-related proteins, such as Mfn2, Fis1, P62, and LC3, as well as Nrf2, Keap1, PINK1, and Parkin, were examined by immunohistochemistry, western blot, and real-time PCR, respectively. Our results indicated that AS II ameliorated albuminuria, renal histopathology, and podocyte foot process effacement and podocyte apoptosis in diabetic rats. AS II also partially restored the renal expression of mitochondrial dynamics-related and autophagy-related proteins, including Mfn2, Fis1, P62, and LC3. AS II also increased the expression of PINK1 and Parkin associated with mitophagy in diabetic rats. Moreover, AS II facilitated antioxidative stress ability via increasing Nrf2 expression and decreasing Keap1 protein level. These results suggested that AS II ameliorated podocyte injury and mitochondrial dysfunction in diabetic rats partly through regulation of Nrf2 and PINK1 pathway. These important findings might provide an innovative therapeutic strategy for the treatment of DN.Astragaloside II (AS II), a novel saponin purified from Astragalus membranes, has been reported to modulate the immune response, repair tissue injury, and prevent inflammatory response. However, the protective effects of AS II on podocyte injury in diabetic nephropathy (DN) have not been investigated yet. In this study, we aimed to investigate the beneficial effects of AS II on podocyte injury and mitochondrial dysfunction in DN. Diabetes was induced with streptozotocin (STZ) by intraperitoneal injection at 55 mg/kg in rats. Diabetic rats were randomly divided into four groups, namely, diabetic rats and diabetic rats treated with losartan (10 mg·kg Diabetic nephrology (DN) is one of the most common causes of end-stage renal disease (ESRD) . PodocytMoreover, increased mitochondrial fragments caused by mitochondrial damage promote autophagic clearance of mitochondria . DysfuncFurthermore, there was a potential relationship between P62 and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid-derived-2-like 2 (Nrf2) pathway that associated with oxidative stress . Nrf2 isAstragalus membranaceus (huang qi) is one of the most widely used ‘‘benefiting vital energy (Yiqi)” herbs in TCM to treat various diseases. Astragalus membranaceus was found to have renal protective effects on DN (Diabetes or diabetic nephrology is considered as “consumptive thirst (Xiaoke)” in Traditional Chinese medicine (TCM) ancient literature. The relevant disease name, symptoms, complications, and treatment of consumptive thirst were recorded in Huangdi’s Internal Classic, the foundation of basic Chinese medical theories . TCM hasts on DN . It has ts on DN . AS II ats on DN . HoweverAS II (with a purity of over 98%) was purchased from Shanghai Jingke Chemical Technology Co., Ltd. , and was suspended in olive oil , as a vehicle for administration to rats. Losartan was purchased from Merck Sharp & Dohme Limited  and suspended in 0.5% methylcellulose solution. Streptozotocin (STZ) was purchased from Sigma-Aldrich Company  and was dissolved in the citrate buffer  .n = 7/each group): 1) STZ-induced diabetic rats (STZ), received an equal volume of olive oil; 2) diabetic rats treated with losartan at 10 mg·kg−1·d−1; 3) diabetic rats treated with low-concentration of AS II at 3.2 mg·kg−1·d−1 (ASIIL); 4) diabetic treated with high-concentration of AS II at 6.4 mg·kg−1·d−1 (ASIIH). Normal rats were chosen as the normal control . All treatment interventions were administrated at two weeks after STZ injection and were administered via oral gavage to rats for nine weeks. Rats were kept in individual metabolic cages for 24 h urine collections at the end of 0 and 9 weeks of treatments. Rats were then anesthetized with pentobarbital sodium and the blood samples were collected through the abdominal aorta for measuring biochemical parameters by using an automatic biochemistry analyzer, and the kidneys were harvested immediately.All the animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals proposed by National Institutes of Health (NIH) and were approved by the Animal Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Healthy 8-week-old male Sprague-Dawley rats weighing 200–250 g were housed under a constant 12 h light–dark cycle at a temperature between 21 and 23°C and allowed free access to food and water. Diabetes was induced in rats by a single intraperitoneal injection of STZ at 55 mg/kg, according to our previous study . SeventyUrine was centrifuged at 3,500 rpm for 10 min at 4°C and the supernatants of urine were measured for the urinary albumin and urinary creatinine by an automatic biochemistry analyzer . Urinary albumin excretion was expressed as urinary albumin/creatinine ratio (ACR). Blood was centrifuged at 3,500 rpm for 10 min at 4°C and the supernatants of blood were measured for blood glucose (GLU) by an automatic biochemistry analyzer .The kidneys were fixed in 10% buffered formalin and embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). The protocol of HE and PAS staining was described previously . Section3 pieces on ice and immediately fixed in 2.5% glutaraldehyde for 2 h at 4°C and washed twice in the same buffer. After dehydration with different concentrations of ethyl alcohol, renal cortices were embedded in epoxy resin 48 h at 60°C. Ultrathin sections were stained with uranyl acetate and lead citrate and examined by TEM. The number of foot processes (FP) in kidney sections was counted by a blinded observer as previously described . Renal cortices were cut into 1 mmescribed .2O2 for 15 min. The sections were blocked by 10% goat serum for 1 h at room temperature. Then, the sections were incubated overnight at 4°C with the following primary antibodies: WT1 , nephrin , cleaved-caspase-3 , p62 , LC3 , Mfn2 , Fis1 , PINK1 , parkin , Nrf2 , and Keap1 . The secondary antibodies  were added to the sections, which were incubated for 1 h at 37°C. Finally, the sections were counterstained using diaminobenzidine and hematoxylin staining and then were rinsed after dehydration. Images were then collected using a light microscope . The positive-staining cells in each kidney section were semiquantified by ImageJ software .Paraffin-embedded kidney tissues were deparaffinized and rehydrated, xylene, ethanol, and quenched by 3% HIn Situ Apoptosis Detection Kit, Merck, S7165). Afterward, the sections were incubated with the primary antibody of WT1 diluted in PBS  at 4°C overnight. Then, secondary antibodies conjugated to Alexa Fluor 488  were added to the sections for 1–2 h and counterstained with DAPI. The triple-positive cells  were identified as the apoptotic podocytes. All fluorescent images were visualized under a confocal microscope  by two blinded investigators.Triple immunofluorescence labeling, including terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, WT1, and 4,6-diamidino-2-phenylindole (DAPI), was used to examine the podocyte apoptosis. TUNEL assay was conducted on frozen sections of kidney samples (4 μm) according to the manufacturer’s instructions  kit assay . The tissue lysates, mixed with an equal amount of 5× SDS loading buffer, were denatured in boiling water for 10 min. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto polyvinylidene difluoride (PVDF) membranes, which were then blocked with Tris-buffered saline Tween-20 (TBST) containing 5% nonfat milk for 1 h at RT. Next, membranes were incubated overnight at 4°C with the following primary antibodies: nephrin , cleaved-caspase-3 , p62 , LC3 , Mfn2 , Fis1 , PINK1 , parkin , Nrf2 , Keap1 , and β-actin . After washing for 5–10 min three times with TBST, membranes were incubated with HRP-bounded secondary antibodies  for 1 h at RT and washed 5–10 min for three times with TBST again. Finally, proteins were visualized by ECL and ImageJ was used to determine band intensity. Each blot was independently repeated three times.−ΔΔct method and normalized to β-actin.Total RNA was extracted from kidney tissues using TRIzol  and reverse-transcribed to cDNA with a Reverse Transcription Kit  following the manufacturer’s protocol. Real-time PCR was performed by Light Cycler 480 system  using SYBR Green Master Mix . The primer sequences are as follows: nephrin: 5′-CGTGCTGGTGATGACTGTACG (forward) and 5′-CGTTCTTGTTCTCCGATTGTG (reverse); cleaved-caspase-3: 5′- CTCGGTCTGGTACAGATGTCG (forward) and 5′-TGGCTCAGAAGCACACAAAC (reverse); Mfn2: 5′-ACCATCAGTAGCCAATCTGGAC (forward) and 5′-AGAGCAGGGACATCTCGTTTC (reverse); Fis1: 5′-CTGGACTCATTGGACTGGCTGTG (forward) and 5′-AGGAAGGCGGTGGTGAGGATG (reverse). The target gene expression was calculated using the 2t-tests and differences among multiple groups were performed using one-way analysis of variance (ANOVA). Data were considered statistically significant if p < 0.05.Data were analyzed using GraphPad Prism software (version 7.0) and presented as means ± standard deviation (SD). Differences between two groups were compared by unpaired Student's To identify the renoprotective effects of AS II in STZ-induced diabetic rats, the levels of blood glucose (GLU) and urinary ACR were examined. Compared with the normal control rats, ACR level at baseline was markedly increased in diabetic rats . HoweverWe performed histological examination of renal tissues from all groups. As shown in HE and PAS staining , diabetiTo further assess the protective effects of AS II on podocytes in diabetic rats, we analyzed the expression of nephrin, which is a podocyte-specific protein and essential for the functional and morphological integrity of glomerular filtration barrier . As showTriple immunofluorescence labeling, including TUNE, WT1, and DAPI, was used to examine the podocyte apoptosis in diabetic rats. Cleaved-caspase-3 (c-caspase-3) is an active form of caspase-3. It is well known for its role in apoptosis pathway, which is related to the deterioration of DN . In thisTo preferably understand the protective effects of AS II on mitochondrial biology, the changes of mitochondrial morphology and dynamics were observed by TEM, immunohistochemistry staining, western blot, and RT-qPCR assays. In Given that Fis1 is essential for mitochondrial fission, while mitochondrial fusion is mediated by Mfn2, we then investigated the expression of Fis1 and Mfn2 to evaluate the effect of AS II on mitochondrial dynamics. The results showed that diabetic rats exhibited a significant reduction of Mfn2 protein, while Fis1 expression was upregulated compared with nondiabetic rats. However, AS II and losartan treatment increased the expression of Mfn2 and conversely decreased the expression of Fis1 in the kidneys of diabetic rats . In summWe further examined the level of autophagy-related proteins in kidney tissues, such as LC3 and P62 proteins, to verify the effect of AS II on autophagy in DN. As is shown in Moreover, the expressions of PINK1 and Parkin related to mitophagy were apparently decreased in diabetic rats, whereas AS II and losartan treatment led to the enhancement of PINK1 and Parkin expression . BecauseAstragalus membranaceus, has been reported to protect against DN in animal models (Astragalosides have a wide range of pharmacological properties, such as anti-inflammatory, antiviral, and immunomodulatory activities . Astragal models . HoweverFirstly, we examined the protective effect of AS II on albuminuria in diabetic rats. According to the recent Secondly, we investigated the beneficial effect of AS II on podocyte injury and mitochondrial dysfunction in diabetic rats. Podocyte apoptosis is considered to be the major mechanism of podocyte loss, which exacerbated renal function decline and proteinuria . PodocytWe next investigated the effects of AS II on autophagy in diabetic rats. Autophagy is a highly regulated lysosomal degradation system . It is aThere is mounting evidence of gender-specific aspects in renal diseases. It was reported that intact males developed proteinuria and kidney injury, whereas females were protected from injury . Both ouAstragalus membranaceus ameliorated albuminuria in diabetic rats and did not cause apparent toxicity to the kidney and liver (Astragalus membranaceus, induced obvious natriuresis and did not markedly affect mean arterial blood pressure, heart rate, and plasma concentration of creatinine in healthy men, suggesting that ARE is a safe natriuretic agent (Our previous study found that nd liver . Our cliic agent . A previic agent . In thisin vivo, and additional studies are needed to determine the underlying mechanism of AS II in vitro. Secondly, we did not investigate the effects of AS II on the normal control group and these effects will be investigated in further study.However, there are limitations in our study. Firstly, we only investigated the effects of AS II Taken together, this study clearly demonstrated that AS II ameliorating podocyte injury and mitochondrial dysfunction in STZ-induced diabetic rats partly through comodulation of Nrf2 and PINK1. These novel findings might pave the way to a novel therapeutic strategy for the treatment of DN.