Non-lethal sonodynamic therapy mitigates hypertensive renal fibrosis through the PI3K/AKT/mTORC1-autophagy pathway.

Hypertension constitutes a significant public health concern, characterized by a high incidence and mortality rate. Hypertensive kidney disease is a prevalent complication associated with hypertension and is the second leading cause of end-stage renal disease (ESRD). Renal fibrosis linked to hypertension has emerged as the third leading cause of disease in dialysis patients. Autophagy activity is crucial for maintaining homeostasis, vitality, and physiological function of kidney cells, while also protecting the kidneys from fibrosis. The deficiency of autophagy will increase the sensitivity of the kidney to the damage, leading to impaired renal function, accumulation of damaged mitochondria and more severe of renal fibrosis. However, enhancing autophagy by activating the PI3K/AKT, AMPK, and mTOR pathways, improves podocyte injury and renal pathological changes, and ameliorates renal function. Current clinical interventions aimed at halting or reversing renal fibrosis in hypertensive patients are notably limited in their efficacy. Here, we present Non-lethal Sonodynamic Therapy (NL-SDT), in which ultrasound is used to activate locally sonosensitizers, thereby stimulating the production of reactive oxygen species for the purpose of modulating cell function or fate, as a novel methodology to inhibit progression of hypertensive renal fibrosis.To confirm whether NL-SDT can reduce hypertensive renal fibrosis and its mechanism. The mice model of hypertensive renal fibrosis was established by using osmotic minipumps (Alzet model 2004, Cupertino, CA) equipped with angiotensin-II (Ang II). The pumps were implanted in mice, ensuring constant infusion of Ang II at a dose of 1.0 µg/kg per minute for 4 weeks. The mice were exposed to 0.4 W/cm2 intensity ultrasonic radiation for 15 min at 4 h post injection of sinoporphyrin sodium (DVDMS) (4 mg/kg) into the caudal vein was repeated weekly for 4 treatments. The kidney from mice was stained with masson's trichrome staining for collagen fiber expression, while alpha-smooth muscle actin (α-SMA) expression was determined via immunohistochemical staining. The protein levels of fibrosis parameters (α-SMA, collagen I, vimentin), pathway-related proteins (PI3K, AKT, mTORC1) and autophagy-related protein LC3B were determined using western blotting. Intracellular reactive oxygen species (ROS) levels were detected using DCFH-DA probe. Immunofluorescence was also used to observe the expression of α-SMA and E-cadherin in cells. Pathway-related protein inhibitors (the autophagy-related inhibitor 3-methyladenine (3-MA), chloroquine (CQ), ROS inhibitor N-acetyl-L-cysteine (NAC) were applied, and autophagosome changes were observed under transmission electron microscopy. Immunofluorescence was used to observe LC3 spot formation within cells.We obtained the following results via animal and cellular research. In vivo, (1) The collagen area of renal tissue was increased significantly in Ang II group (50.6%). The positive expression of α-SMA was increased significantly (37.8%). (2) The collagen area decreased after NL-SDT treatment (34.8%). The expression of α-SMA was decreased too (48.9%). The expression of LC3B increased in NL-SDT group. (3) The effect of NL-SDT on reducing renal fibrosis can be changed by rapamycin and CQ. In vitro. (1) The expression of α-SMA, collagen I and vimentin were increased significantly in TGF-β1-induced NRK-52E cells. (2) The increase of autophagosomes was observed in TGF-β1-induced NRK-52E cells after NL-SDT. The levels of ROS were increased after NL-SDT (24.8%). The effect of NL-SDT on autophagy was reversed after administration of NAC. The expression of PI3K, P-AKT and P-mTORC1 was decreased in TGF-β1-induced NRK-52E cells after NL-SDT. NL-SDT inhibited the transition of epithelial cells into myofibroblasts by activating PI3K-AKT-mTORC1-autophagy pathway in TGF-β1-induced NRK-52E cells. (3) The administration of the pathway inhibitors showed a reciprocal effect on NL-SDT-inhibited epithelial-mesenchymal transition (EMT).(1) NL-SDT reduced blood pressure temporarily in mice model of hypertensive renal fibrosis induced by Ang II. (2) NL-SDT alleviated renal fibrosis in mice model of hypertensive renal fibrosis induced by Ang II. (3) NL-SDT promoted autophagy by inhibiting PI3K-AKT-mTORC1 signaling pathway and alleviated renal fibrosis in mice model of hypertensive renal fibrosis induced by Ang II. NL-SDT is a non-invasive and efficacious regimen to inhibit renal fibrosis. It may be a new approach for clinical treatment of renal fibrosis, delaying or reducing the occurrence of ESRD.

Liu D ，Wang H ，Li J ，Sheng S ，Wang S ，Tian Y ... -  《Scientific Reports》

LYC inhibits the AKT signaling pathway to activate autophagy and ameliorate TGFB-induced renal fibrosis.

Wang Y ，Ping Z ，Gao H ，Liu Z ，Xv Q ，Jiang X ，Yu W ... -  《-》

Danggui Buxue decoction regulates autophagy to Improve renal fibrosis in diabetes through miR-27a /PI3K/AKT pathway.

Danggui Buxue Decoction (DBD) is a classic traditional Chinese herbal formulation, composed of Astragali Radix (AR) and Angelica Sinensis Radix (ASR) in a ratio of 5:1. It is a traditional Chinese medicine classic prescription for nourishing Qi and Yin (vital energy and body fluids), and it is effective in treating various clinical diseases. Diabetic nephropathy (DN) is categorized under "thirsting," "edema," and "turbid urine" in Traditional Chinese Medicine (TCM). However, the underlying mechanisms by which DBD ameliorates diabetic nephropathy remain unclear. To explore the mechanism by which Danggui Buxue Decoction (DBD) regulates podocyte autophagy in diabetic nephropathy (DN). Male db/m mice served as controls; db/db mice were divided into the model, dapagliflozin, and high/low-dose DBD groups. After 12 weeks of gavage, body weight, fasting blood glucose, urine albumin-to-creatinine ratio, 24-h urine volume, and blood urea nitrogen were recorded. Renal autophagy was assessed by Masson staining; mRNA levels were measured by qRT-PCR; and protein expression was analyzed by Western blot. The expression of inflammatory factors in the kidney was measured by ELISA. Human renal podocytes were cultured in NG, HG, HG + Blank serum, and HG + DBD-containing serum groups for 48 h; cell viability was measured by CCK-8, and autophagy was observed by transmission electron microscopy. Changes in autophagy protein and mRNA expression were observed after miR-27a transfection under high glucose conditions. DBD can ameliorate renal function and reduce the degree of renal fibrosis in DN mice, enhance the mRNA expression of Beclin-1 and ULK1, and decrease the mRNA expression of Vimentin and α-SMA. This trend mirrors protein expression, and DBD also lowers renal inflammatory factors. DBD-containing serum boosts human renal podocyte viability under high glucose, protecting cells and modulating mRNA levels of Beclin-1, ULK1, P62, and PI3K, with miR-27a-mimic reversing these effects. DBD-containing serum also enhances Beclin-1, suppresses P62, and reduces the expression of p-PI3K/PI3K and p-AKT/AKT. DBD Regulates Autophagy to Improve Renal Fibrosis in Diabetes via the miR-27a/PI3K/AKT Pathway.

Wang N ，Feng H ，Zhang Z ，Tian H ，Gu L ，Bian Y ，Xue M ... -  《-》

Rg1 improves Alzheimer's disease by regulating mitochondrial dynamics mediated by the AMPK/Drp1 signaling pathway.

Alzheimer's disease (AD) is the most prevalent form of dementia, characterized by a complex pathogenesis that includes Aβ deposition, abnormal phosphorylation of tau protein, chronic neuroinflammation, and mitochondrial dysfunction. In traditional medicine, ginseng is revered as the 'king of herbs'. Ginseng has the effects of greatly tonifying vital energy, strengthening the spleen and benefiting the lungs, generating fluids and nourishing the blood, and calming the mind while enhancing intelligence. Ginsenoside Rg1 (Rg1) is a well-defined major active component found in ginseng, known for its relatively high content. It has been demonstrated to exhibit neuroprotective effects in both in vivo and in vitro models, capable of ameliorating Aβ and tau pathology, regulating synaptic function, and reducing inflammation, oxidative stress, and apoptosis. However, the potential of Rg1 to improve AD pathology through the regulation of mitochondrial dynamics is still uncertain. Despite the active research efforts on drugs for AD, the currently available anti-AD medications can only slow disease progression and manage symptoms, yet unable to provide a cure for AD. Furthermore, some anti-AD drugs failed phase III and IV clinical trials due to significant side effects. Therefore, there is an urgent need to further investigate the pathogenesis of AD, to identify new therapeutic targets, and to explore more effective therapies. The aim of this study is to evaluate the potential therapeutic effects of Rg1 on APP/PS1 double transgenic mice and Aβ42-induced HT22 cell models, and to investigate the potential mechanisms through which it provides neuroprotective effects. This study investigates the effects of Rg1 in treating AD on APP/PS1 double transgenic mice and Aβ42-induced HT22 cells. In the in vivo experiments, APP/PS1 mice were divided into a model group, Rg1-L group, Rg1-H group, and donepezil group, with C57BL/6 mice serving as the control group (n = 12 per group). The Rg1-L and Rg1-H groups were administered Rg1 at doses of 5 mg/kg/d and 10 mg/kg/d, respectively, while the donepezil group received donepezil at a dose of 1.3 mg/kg/d. Both the control and model groups received an equal volume of physiological saline daily for 28 days. Learning and spatial memory were assessed by the Morris water maze (MWM) and novel object recognition (NOR) tests, and neuronal damage by Nissl staining. Aβ deposition was analyzed through immunohistochemistry and Western blot, while the expression levels of synaptic proteins PSD95 and SYN were evaluated via immunofluorescence staining and Western blot. The dendritic spines of neurons was observed by Golgi staining.The ultrastructure of neuronal mitochondria and synapses was examined by transmission electron microscopy (TEM). Mitochondrial function was assessed through measurements of Reactive oxygen species (ROS), Superoxide Dismutase (SOD), and Adenosine Triphosphate (ATP), and Western blot analysis was performed to detect the expression levels of AMPK, p-AMPK, Drp1, p-Drp1, OPA1, Mfn1, and Mfn2, thereby investigating the protective effects of Rg1 on mitochondrial dysfunction and cognitive impairment in APP/PS1 double transgenic mice. In vitro experiments, HT22 cells were treated with Aβ42 of 10 μM for 24 h to verify the therapeutic effects of Rg1. Flow cytometry was used to detect ROS and JC-1, biochemical methods were employed to measure SOD and ATP, immunofluorescence staining was used to detect the expression levels of PSD95 and SYN, and Western blot analysis was conducted to elucidate its potential mechanisms of action. The findings suggest that after 28 days of Rg1 treatment, cognitive dysfunction in APP/PS1 mice was improved. Pathological and immunohistochemical analyses demonstrated that Rg1 treatment significantly reduced Aβ deposition and neuronal loss. Rg1 can improve synaptic dysfunction and mitochondrial function in APP/PS1 mice. Rg1 activated AMPK, enhanced p-AMPK expression, inhibited Drp1, and reduced p-Drp1 levels, which led to increased expression of OPA1, Mfn1, and Mfn2, thereby inhibiting mitochondrial fission and facilitating mitochondrial fusion. Additionally, Rg1 effectively reversed the decrease in mitochondrial membrane potential (MMP) and the increase in ROS production induced by Aβ42 in HT22 cells, restoring SOD and ATP levels. Furthermore, Rg1 regulated mitochondrial fission mediated by the AMPK/Drp1 signaling pathway, promoting mitochondrial fusion and improving synaptic dysfunction. Our research provides evidence for the neuroprotective mechanisms of Rg1 in AD models. Rg1 modulates mitochondrial dynamics through the AMPK/Drp1 signaling pathway, thereby reducing synaptic and mitochondrial dysfunction in APP/PS1 mice and AD cell models.

Zhang Y ，Liu S ，Cao D ，Zhao M ，Lu H ，Wang P ... -  《-》

Rapamycin prevents cyclophosphamide-induced ovarian follicular loss and potentially inhibits tumour proliferation in a breast cancer xenograft mouse model.

To what extent and via what mechanism does the concomitant administration of rapamycin (a follicle activation pathway inhibitor and antitumour agent) and cyclophosphamide (a highly toxic ovarian anticancer agent) prevent cyclophosphamide-induced ovarian reserve loss and inhibit tumour proliferation in a breast cancer xenograft mouse model? Daily concomitant administration of rapamycin and a cyclic regimen of cyclophosphamide, which has sufficient antitumour effects as a single agent, suppressed cyclophosphamide-induced primordial follicle loss by inhibiting primordial follicle activation in a breast cancer xenograft mouse model, suggesting the potential of an additive inhibitory effect against tumour proliferation. Cyclophosphamide stimulates primordial follicles by activating the mammalian target of the rapamycin (mTOR) pathway, resulting in the accumulation of primary follicles, most of which undergo apoptosis. Rapamycin, an mTOR inhibitor, regulates primordial follicle activation and exhibits potential inhibitory effects against breast cancer cell proliferation. To assess ovarian follicular apoptosis, 3 weeks after administering breast cancer cells, 8-week-old mice were randomized into three treatment groups: control, cyclophosphamide, and cyclophosphamide + rapamycin (Cy + Rap) (n = 5 or 6 mice/group). Mice were treated with rapamycin or vehicle control for 1 week, followed by a single dose of cyclophosphamide or vehicle control. Subsequently, the ovaries were resected 24 h after cyclophosphamide administration (short-term treatment groups). To evaluate follicle abundance and the mTOR pathway in ovaries, as well as the antitumour effects and impact on the mTOR pathway in tumours, 8-week-old xenograft breast cancer transplanted mice were randomized into three treatment groups: vehicle control, Cy, and Cy + Rap (n = 6 or 7 mice/group). Rapamycin (5 mg/kg) or the vehicle was administered daily for 29 days. Cyclophosphamide (120 mg/kg) or the vehicle was administered thrice weekly (long-term treatment groups). The tumour diameter was measured weekly. Seven days after the last cyclophosphamide treatment, the ovaries were harvested, fixed, and sectioned (for follicle counting) or frozen (for further analysis). Similarly, the tumours were resected and fixed or frozen. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed to examine ovarian follicular apoptosis in the short-term treatment groups. All subsequent experiments were conducted in the long-term treatment groups. Tumour growth was evaluated using the tumour volume index. The tumour volume index indicates the relative volume, compared to the volume 3 weeks after tumour cell injection (at treatment initiation) set to 100%. Tumour cell proliferation was evaluated by Ki-67 immunostaining. Activation of the mTOR pathway in tumours was assessed using the protein extracts from tumours and analysed by western blotting. Haematoxylin and eosin staining of ovaries was used to perform differential follicle counts for primordial, primary, secondary, antral, and atretic follicles. Activation of the mTOR pathway in ovaries was assessed using protein extracts from whole ovaries and analysed by western blotting. Localization of mTOR pathway activation within ovaries was assessed by performing anti-phospho-S6 kinase (downstream of mTOR pathway) immunohistochemistry. Ovaries of the short-term treatment groups were resected 24 h after cyclophosphamide administration and subjected to TUNEL staining of apoptotic cells. No TUNEL-positive primordial follicles were detected in the control, Cy, and Cy + Rap groups. Conversely, many granulosa cells of growing follicles were TUNEL positive in the Cy group but negative in the control and Cy + Rap groups. All subsequent experimental results were obtained from the long-term treatment groups. The tumour volume index stabilized at a mean of 160-200% in the Cy group and 130% in the Cy + Rap group throughout the treatment period. In contrast, tumours in the vehicle control group grew continuously with a mean tumour volume index of 600%, significantly greater than that of the two treatment groups. Based on the western blot analysis of tumours, the mTOR pathway was activated in the vehicle control group and downregulated in the Cy + Rap group when compared with the control and Cy groups. Ki-67 immunostaining of tumours showed significant inhibition of cell proliferation in the Cy + Rap group when compared with that in the control and Cy groups. The ovarian follicle count revealed that the Cy group had significantly fewer primordial follicles (P < 0.001) than the control group, whereas the Cy + Rap group had significantly higher number of primordial follicles (P < 0.001, 2.5 times) than the Cy group. The ratio of primary to primordial follicles was twice as high in the Cy group than in the control group; however, no significant difference was observed between the control group and the Cy + Rap group. Western blot analysis of ovaries revealed that the mTOR pathway was activated by cyclophosphamide and inhibited by rapamycin. The phospho-S6 kinase (pS6K)-positive primordial follicle rate was 2.7 times higher in the Cy group than in the control group. However, this effect was suppressed to a level similar to the control group in the Cy + Rap group. None. The combinatorial treatment of breast cancer tumours with rapamycin and cyclophosphamide elicited inhibitory effects on cell proliferative potential compared to cyclophosphamide monotherapy. However, no statistically significant additive effect was observed on tumour volume. Thus, the beneficial antitumour effect afforded by rapamycin administration on breast cancer could not be definitively proven. Although rapamycin has ovarian-protective effects, it does not fully counteract the ovarian toxicity of cyclophosphamide. Nevertheless, rapamycin is advantageous as an ovarian protective agent as it can be used in combination with other ovarian protective agents, such as hormonal therapy. Hence, in combination with other agents, mTOR inhibitors may be sufficiently ovario-protective against high-dose and cyclic cyclophosphamide regimens. Compared with a cyclic cyclophosphamide regimen that replicates human clinical practice under breast cancer-bearing conditions, the combination with rapamycin mitigates the ovarian follicle loss of cyclophosphamide without interfering with the anticipated antitumour effects. Hence, rapamycin may represent a new non-invasive treatment option for cyclophosphamide-induced ovarian dysfunction in breast cancer patients. This work was not financially supported. The authors declare that they have no conflict of interest.

Tanaka Y ，Amano T ，Nakamura A ，Yoshino F ，Takebayashi A ，Takahashi A ，Yamanaka H ，Inatomi A ，Hanada T ，Yoneoka Y ，Tsuji S ，Murakami T ... -  《-》