Btg2 Promotes Focal Segmental Glomerulosclerosis via Smad3-Dependent Podocyte-Mesenchymal Transition.

Podocyte injury plays a critical role in the progression of focal segmental glomerulosclerosis (FSGS). Here, it is reported that B-cell translocation gene 2 (Btg2) promotes Adriamycin (ADR)-induced FSGS via Smad3-dependent podocyte-mesenchymal transition. It is found that in FSGS patients and animal models, Btg2 is markedly upregulated by podocytes and correlated with progressive renal injury. Podocyte-specific deletion of Btg2 protected against the onset of proteinuria and glomerulosclerosis in ADR-treated mice along with inhibition of EMT markers such as α-SMA and vimentin while restoring epithelial marker E-cadherin. In cultured MPC5 podocytes, overexpression of Btg2 largely promoted ADR and TGF-ß1-induced EMT and fibrosis, which is further enhanced by overexpressing Btg2 but blocked by disrupting Btg2. Mechanistically, Btg2 is rapidly induced by TGF-ß1 and then bound Smad3 but not Smad2 to promote Smad3 signaling and podocyte EMT, which is again exacerbated by overexpressing Btg2 but blocked by deleting Btg2 in MPC5 podocytes. Interestingly, blockade of Smad3 signaling with a Smad3 inhibitor SIS3 is also capable of inhibiting Btg2 expression and Btg2-mediated podocyte EMT, revealing a TGF-ß/Smad3-Btg2 circuit mechanism in Btg2-mediated podocyte injury in FSGS. In conclusion, Btg2 is pathogenic in FSGS and promotes podocyte injury via a Smad3-dependent EMT pathway.

Synergism of calycosin and bone marrow-derived mesenchymal stem cells to combat podocyte apoptosis to alleviate adriamycin-induced focal segmental glomerulosclerosis.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (MSCs) show podocyte-protective effects in chronic kidney disease. Calycosin (CA), a phytoestrogen, is isolated from Astragalus membranaceus with a kidney-tonifying effect. CA preconditioning enhances the protective effect of MSCs against renal fibrosis in mice with unilateral ureteral occlusion. However, the protective effect and underlying mechanism of CA-pretreated MSCs (MSCsCA) on podocytes in adriamycin (ADR)-induced focal segmental glomerulosclerosis (FSGS) mice remain unclear. AIM: To investigate whether CA enhances the role of MSCs in protecting against podocyte injury induced by ADR and the possible mechanism involved. METHODS: ADR was used to induce FSGS in mice, and MSCs, CA, or MSCsCA were administered to mice. Their protective effect and possible mechanism of action on podocytes were observed by Western blot, immunohistochemistry, immunofluorescence, and real-time polymerase chain reaction. In vitro, ADR was used to stimulate mouse podocytes (MPC5) to induce injury, and the supernatants from MSC-, CA-, or MSCsCA-treated cells were collected to observe their protective effects on podocytes. Subsequently, the apoptosis of podocytes was detected in vivo and in vitro by Western blot, TUNEL assay, and immunofluorescence. Overexpression of Smad3, which is involved in apoptosis, was then induced to evaluate whether the MSCsCA-mediated podocyte protective effect is associated with Smad3 inhibition in MPC5 cells. RESULTS: CA-pretreated MSCs enhanced the protective effect of MSCs against podocyte injury and the ability to inhibit podocyte apoptosis in ADR-induced FSGS mice and MPC5 cells. Expression of p-Smad3 was upregulated in mice with ADR-induced FSGS and MPC5 cells, which was reversed by MSCCA treatment more significantly than by MSCs or CA alone. When Smad3 was overexpressed in MPC5 cells, MSCsCA could not fulfill their potential to inhibit podocyte apoptosis. CONCLUSION: MSCsCA enhance the protection of MSCs against ADR-induced podocyte apoptosis. The underlying mechanism may be related to MSCsCA-targeted inhibition of p-Smad3 in podocytes.

Hederagenin ameliorates renal fibrosis in chronic kidney disease through blocking ISG15 regulated JAK/STAT signaling.

Interstitial fibrosis is the key pathological characteristics of chronic kidney diseases (CKD). In this study, we reported that hederagenin (HDG) can effectively improve the renal interstitial fibrosis and its mechanism. We constructed CKD animal models of ischemia reperfusion injury (IRI) and unilateral ureteral obstruction (UUO) respectively to observe the improvement effect of HDG on CKD. The results showed that HDG can effectively improve the pathological structure of kidney and the renal fibrosis in CKD mice. Meanwhile, HDG can also significantly reduce the expression of α-SMA and FN induced by TGF-ß in Transformed C3H Mouse Kidney-1 (TCMK1) cells. Mechanistically, we performed transcriptome sequencing on UUO kidneys treated with HDG. By real time PCR screening of the sequencing results, we determined that ISG15 plays an important role in the intervention of HDG in CKD. Subsequently, we knocked-down ISG15 in TCMK1 and found that ISG15 knock-down significantly inhibited TGF-ß-induced fibrotic protein expression and JAK/STAT activation. Finally, we performed electrotransfection and used liposomes to transfect ISG15 overexpression plasmids to up-regulate ISG15 in kidney and cells, respectively. We found that ISG15 can aggravate renal tubular cell fibrosis and abolish the protection of HDG on CKD. These results indicated that HDG significantly improves renal fibrosis in CKD by inhibiting ISG15 and its downstream JAK/STAT signaling pathway, which provides a new drug and research target for the subsequent treatment of CKD.

Macrophages mediate psoriasis via Mincle-dependent mechanism in mice.

Psoriasis is currently considered to be an immune and inflammatory disease characterized by massive immune cells infiltration including macrophages. It has been reported that macrophage-inducible C-type lectin (Mincle) is essential to maintain the pro-inflammatory phenotype of M1 macrophages, however, its role and mechanisms in psoriasis remain largely unknown. A model of psoriasis was induced in mice by a daily topical application of imiquimod for 7 days. Role and mechanisms of Mincle in macrophage-mediated psoriasis were investigated in clodronate liposomes induced macrophage depletion mice followed by adoptively transferring with Mincle-expressing or -knockout (KO) macrophages, and in macrophage specific Mincle knockout mice (Mincleloxp/loxp/Lyz2-cre+/+). Finally, a Mincle neutralizing antibody was employed to the psoriasis mice to reveal the therapeutic potential for psoriasis by targeting Mincle. Mincle was highly expressed by M1 macrophages in the skin lesions of patients and mice with psoriasis. Clodronate liposomes-induced macrophage depletion inhibited psoriasis in mice, which was restored by adoptive transfer with Mincle-expressing macrophages but not by Mincle-KO macrophages. This was further confirmed in macrophage-specific Mincle-KO mice. Mechanistically, macrophages mediated psoriasis via the Mincle-Syk-NF-κB pathway as blocking macrophage Mincle inhibited Syk/NF-κB-driven skin lesions and epidermal injury in vivo and in vitro. We also found that LPS induced Mincle expression by M1 macrophages via the PU.1-dependent mechanism. Most importantly, we revealed that targeting Mincle with a neutralizing antibody significantly improved psoriasis in mice. In summary, our findings demonstrated that macrophages mediate psoriasis in mice via the Mincle-dependent mechanism, targeting Mincle may represent as a novel therapy for psoriasis. A simplified pathway model of Mincle in macrophage-mediated psoriasis.

Neuropeptide Y protects kidney from acute kidney injury by inactivating M1 macrophages via the Y1R-NF-κB-Mincle-dependent mechanism.

Neuropeptide Y (NPY) is produced by the nerve system and may contribute to the progression of CKD. The present study found the new protective role for NPY in AKI in both patients and animal models. Interestingly, NPY was constitutively expressed in blood and resident kidney macrophages by co-expressing NPY and CD68+ markers, which was lost in patients and mice with AKI-induced by cisplatin. Unexpectedly, NPY was renoprotective in AKI as mice lacking NPY developed worse renal necroinflammation and renal dysfunction in cisplatin and ischemic-induced AKI. Importantly, NPY was also a therapeutic agent for AKI because treatment with exogenous NPY dose-dependently inhibited cisplatin-induced AKI. Mechanistically, NPY protected kidney from AKI by inactivating M1 macrophages via the Y1R-NF-κB-Mincle-dependent mechanism as deleting or silencing NPY decreased Y1R but increased NF-κB-Mincle-mediated M1macrophage activation and renal necroinflammation, which were reversed by addition of NPY or by silencing Mincle but promoted by blocking Y1R with BIBP 3226. Thus, NPY is renoprotective and may be a novel therapeutic agent for AKI. NPY may act via Y1R to protect kidney from AKI by blocking NF-κB-Mincle-mediated M1 macrophage activation and renal necroinflammation.

Calycosin pretreatment enhanced the therapeutic efficacy of mesenchymal stem cells to alleviate unilateral ureteral obstruction-induced renal fibrosis by inhibiting necroptosis.

Bone marrow-derived mesenchymal stem cells (MSCs) show antifibrotic activity in various chronic kidney diseases. Here, we aimed to investigate whether Calycosin (CA), a phytoestrogen, could enhance the antifibrotic activity of MSCs in primary tubular epithelial cells (PTECs) induced by TGF-ß1 and in a mouse model of unilateral ureteral obstruction (UUO). We found that MSCs treatment significantly inhibited fibrosis, and CA pretreatment enhanced the effects of MSCs on fibrosis in vitro. Consistent with the in vitro studies, MSCs alleviated tubular injury and renal fibrosis in mice after UUO, and CA-pretreated MSCs resulted in more significant improvements in tubular injury and renal fibrosis than MSCs after UUO. Moreover, MSCs treatment significantly inhibited necroptosis by repressing the elevation of MLKL, RIPK1, and RIPK3 in PTECs treated by TGF-ß1and in mice after UUO, and CA-pretreated MSCs were superior to MSCs in alleviating necroptosis. MSCs significantly reduced TNF-α and TNFR1 expression induced by TGF-ß1 in PTECs and inhibited TGF-ß1, TNF-α, and TNFR1 expression induced by UUO in mice. These effects of MSCs were significantly enhanced after CA pretreatment. Therefore, our results suggest that CA pretreatment enhances the antifibrotic activity of MSCs by inhibiting TGF-ß1/TNF-α/TNFR1 signaling-induced necroptosis.

Huanglian Jiedu plaster ameliorated X-ray-induced radiation dermatitis injury by inhibiting HMGB1-mediated macrophage-inflammatory interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Huanglian Jiedu plaster (HJP) is a kind of Chinese patent medicine that contains four medicinal plants. It has been clinically proven to be beneficial for the treatment of tumor-associated radiation dermatitis. However, the underlying mechanism of HJP on radiation dermatitis remains unclear. AIM OF THE STUDY: This study aims to investigate the therapeutic effect of HJP on X-ray-induced radiation dermatitis, and how HJP improves the inflammatory response and skin damage of radiation dermatitis. MATERIALS AND METHODS: In this study, We selected a case of esophageal cancer as a clinical demonstration of the efficacy of radiation dermatitis. The patient received a total radiation dose of 7000cGY, and treatment by HJP for 14 days.RD mouse models were established through continuous irradiation with X-ray (800cGY) on the right hind limb of mice for 5 days, and the treatment group mice was applied HJP to the irradiated skin for 15 days from modeling. An inflammatory cellular model was induced through irradiation with X-ray (100cGY) in JB6 cells and a co-culture system of JB6 cell and macrophage was established to examine the effect and mechanism of HJP on the inflammatory interaction of these two cells. The activation of HMGB1-TLR4-NF-κB signaling pathway, and the levels of epidermal injury related factors and inflammatory cytokins were subsequently detected. RESULTS: The results showed that HJP can significantly alleviate X-ray-induced skin injury, inhibiting skin inflammation and reducing the expression of inflammatory cytokins (IL-1ß, IL-6, TNF-α) and epidermal damage related factors (Integrin ß1, CXCL9 and Cytokeratin17), as well as significantly down-regulated the protein level of HMGB1 (a key DAMPs factor) in vivo and in vitro. Cell co-culture experiments demonstrated that HMGB1 released from X-ray-induced JB6 cells can promote inflammatory response of macrophage, which then feedback aggravate epithelial cell damage, notably, HJP can significantly improve radiation skin lesion by inhibiting HMGB1-mediated inflammatory interaction between epithelial cells and macrophages. CONCLUSION: In summary, these findings indicated the role of HJP in the treatment of RD by inhibiting the inflammatory interaction between macrophage and JB6 cells mediated by HMGB1, which may provide a reliable therapeutic method for RD. Furthermore, HMGB1 may be an effective target for HJP to inhibit inflammation and ameliorate skin damage in RD.

Hederagenin ameliorates cisplatin-induced acute kidney injury via inhibiting long non-coding RNA A330074k22Rik/Axin2/ß-catenin signalling pathway.

BACKGROUND: Acute kidney injury (AKI), a kidney disease with high morbidity and mortality, is characterized by a dramatic decline in renal function. Hederagenin (HDG), a pentacyclic triterpenoid saponin isolated from astragalus membranaceus, has been shown to have significant anti-inflammatory effects on various diseases. However, the effects of HDG on renal injury and inflammation in AKI has not been elucidated. METHODS: In this research, mice model of AKI was established by intraperitoneal injection of cisplatin in vivo, the inflammatory model of renal tubular epithelial cells was established by LPS stimulation in vitro, and HDG was used to intervene in vitro and in vivo models. Transcriptome sequencing was used to analyze the alterations of LncRNA and mRNA expression in AKI model and LncRNA-A330074k22Rik (A33) knockdown cells, respectively. Renal in situ electrotransfer knockdown plasmid was used to establish mice model of AKI with low expression of A33 in kidney. RESULTS: The results showed that HDG effectively alleviate cisplatin-induced kidney injury and inflammation in mice. Transcriptome sequencing results showed that multiple LncRNAs in kidney of AKI model exhibited significant changes, among which LncRNA-A33 had the most obvious change trend. Subsequent results showed that A33 was highly expressed in kidney of AKI mice and LPS-induced renal tubular cells. After in situ renal electroporation knockdown plasmid down-regulated A33 in kidney of AKI mice, it was found that inhibition of A33 could significantly relieve cisplatin-induced kidney injury and inflammation of AKI, while HDG could effectively suppress the expression of A33 in vitro and in vivo, respectively. Subsequently, transcriptome sequencing was again used to analyze the changes in mRNA expression of renal tubular cells after A33 knockdown by siRNA. The results showed that a large number of inflammation-related signaling pathways were down-regulated, Axin2 and its downstream ß-catenin signal were significantly inhibited. Cell recovery test showed that HDG inhibited Axin2/ß-catenin signal by down-regulating A33, and improved kidney injury and inflammation of AKI. CONCLUSION: Taken together, HDG significantly ameliorated cisplatin-induced kidney injury through LncRNA-A330074k22Rik/Axin2/ß-catenin signal axis, which providing a potential therapeutic approach for the treatment of AKI.

Zhilong Huoxue Tongyu Capsules Ameliorate Early Brain Inflammatory Injury Induced by Intracerebral Hemorrhage via Inhibition of Canonical NFÐºß Signalling Pathway.

Background: Intracerebral hemorrhage (ICH) is a debilitating and fatal condition with continuously rising incidence globally, without effective treatment available. Zhilong Huoxue Tongyu (ZLHXTY) capsule is a traditional Chinese medicine that is used for ICH treatment in China. However, the evidence based mechanism is not clear. Purpose: To study the protective effects of ZLHXTY capsules against ICH pathogenesis via targetting nuclear factor kappa ß (NFкß) canonical signalling pathway. Methods: C57BL/6 J mice ICH models using autologous blood injection were used to study the effect of ZLHXTY (1.4 g/kg P.O.) after 24 and 72 hrs of ICH induction. The neurological scoring, corner turn test and balance beam with scoring was performed to assess neurological damage. Hematoxylin/eosin and nissl staining was used for histopathological evaluation. Levels of TNFα, NFкB, iNOS, COX2, IL1, IL6 were measured using real time qPCR and western blotting. Protein levels of IKKß and IкBα were analyzed through western blotting. Immunofluorescence for co-expression of NeuN/TNFα, NeuN/NFкB, Iba1/TNFα, and Iba1/NFкB was also performed. Results: Treatment with ZLHXTY capsules after ICH ameliorated inflammatory brain injury after 24 and 72 h; revealed by neurological scoring, hematoxylin/eosin and nissl staining. The qPCR and western blot analyses demonstrated significant downregulation of TNFα, NFкB, iNOS, COX2, IL1ß and IL6. Further, the IKKß and IкBα revealed significant downregulation and upregulation respectively in western blot. Immunofluorescence also revealed attenuated expression of TNFα and NFкB in neurons and also low expression of Iba1. Conclusion: ZLHXTY capsules elicit its neuroprotective effect by targetting the NFÐºß canonical signalling pathway, thereby ameliorating the ICH induced brain injury.

Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) improves renal mesangial cell damage in diabetic nephropathy by inhibiting the inflammatory response of infiltrated macrophages.

BACKGROUND: Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease with scantly effective treatment. Numerous evidences indicated that macrophages play an important role in the occurrence and pathogenesis of DN by secreting inflammatory cytokines. Mincle is mainly expressed in macrophages and promotes kidney inflammation and damage of acute kidney injury. However, the role of Mincle in DN is unclear. In this study, we aim to investigate the effect of Mincle-related macrophage inflammation on DN, and whether it can be identified as the therapeutic target for Astragalus mongholicus Bunge and Panax notoginseng Formula (A&P), a widely used Chinese herbal decoction for DN treatment. METHODS: In vivo experiments high-fat and high-sugar diet and streptozotocin was used to establish a diabetic nephropathy model, while in vitro experiments inflammation model was induced by high-glucose in mouse Bone Marrow-Derived Macrophages (BMDM) cells and mouse mesangial (MES) cells. Kidney pathological staining is used to detect kidney tissue damage and inflammation, Western blotting, Real-time PCR and ELISA are performed to detect Mincle signaling pathway related proteins and inflammatory cytokines. RESULTS: Mincle was mainly expressed in infiltrated macrophage of DN kidney, and was significant decreased after A&P administration. The in vitro experiments also proved that A&P effectively down-regulated the expression of Mincle in macrophage stimulated by high glucose. Meanwhile, the data demonstrated that A&P can reduce the activation of NFκB, and the expression and secretion of inflammatory cytokines in DN kidney or BMDM cells. Notably, we set up a co-culture system to conform that BMDM cells can aggravate the inflammatory response of mesangial (MES) cells under high glucose stimulation. Furthermore, we found that the anti-injury role of A&P in MES cells was dependent on inhibition of the Mincle in macrophage. CONCLUSION: In summary, our study found that A&P is effective in reducing renal pathological damage and improving renal function and inflammation in diabetic nephropathy by a mechanism mainly related to the inhibition of the Mincle/Card9/NFκB signaling pathway.

Smad3 deficiency improves islet-based therapy for diabetes and diabetic kidney injury by promoting ß cell proliferation via the E2F3-dependent mechanism.

Rationale: Poor ß cell proliferation is one of the detrimental factors hindering islet cell replacement therapy for patients with diabetes. Smad3 is an important transcriptional factor of TGF-ß signaling and has been shown to promote diabetes by inhibiting ß cell proliferation. Therefore, we hypothesize that Smad3-deficient islets may be a novel cell replacement therapy for diabetes. Methods: We examined this hypothesis in streptozocin-induced type-1 diabetic mice and type-2 diabetic db/db mice by transplanting Smad3 knockout (KO) and wild type (WT) islets under the renal capsule, respectively. The effects of Smad3KO versus WT islet replacement therapy on diabetes and diabetic kidney injury were examined. In addition, RNA-seq was applied to identify the downstream target gene underlying Smad3-regulated ß cell proliferation in Smad3KO-db/db versus Smad3WT-db/db mouse islets. Results: Compared to Smad3WT islet therapy, treatment with Smad3KO islets produced a much better therapeutic effect on both type-1 and type-2 diabetes by significantly lowering serum levels of blood glucose and HbA1c and protected against diabetic kidney injuries by preventing an increase in serum creatinine and the development of proteinuria, mesangial matrix expansion, and fibrosis. These were associated with a significant increase in grafted ß cell proliferation and blood insulin levels, resulting in improved glucose intolerance. Mechanistically, RNA-seq revealed that compared with Smad3WT-db/db mouse islets, deletion of Smad3 from db/db mouse islets markedly upregulated E2F3, a pivotal regulator of cell cycle G1/S entry. Further studies found that Smad3 could bind to the promoter of E2F3, and thus inhibit ß cell proliferation via an E2F3-dependent mechanism as silencing E2F3 abrogated the proliferative effect on Smad3KO ß cells. Conclusion: Smad3-deficient islet replacement therapy can significantly improve both type-1 and type-2 diabetes and protect against diabetic kidney injury, which is mediated by a novel mechanism of E2F3-dependent ß cell proliferation.

LncRNA GAS5 protects against TGF-ß-induced renal fibrosis via the Smad3/miRNA-142-5p axis.

Increasing evidence shows that long noncoding RNAs (lncRNAs) play an important role in kidney disease. In this study, we investigated the role of the lncRNA growth arrest-specific 5 (GAS5) in the pathogenesis of renal fibrosis. We found that GAS5 was markedly decreased in the fibrotic kidney of a unilateral ureteral obstructive nephropathy mouse model. In addition, GAS5 was expressed in mouse tubular epithelial cells (mTECs) and interstitial fibroblasts in normal renal tissue and was especially highly expressed in the cytoplasm. In vitro experiments showed that GAS5 was downregulated by transforming growth factor-ß1 (TGF-ß1) in a dose- and time-dependent manner. Overexpression of GAS5 blocked TGF-ß1-induced collagen type I and fibronectin expression and vice versa. Mechanistic experiments revealed that Smad3 but not Smad2 drove the regulation of GAS5. More importantly, GAS5 interacted with miR-142-5p and was involved in the renoprotective effect by participating in the competing endogenous RNA network. Finally, we also found that knockdown of GAS5 promoted TGF-ß1-induced mouse tubular epithelial cell apoptosis via the Smad3 pathway. Taken together, our results uncovered a lncRNA/miRNA competing endogenous RNA network-based mechanism that modulates extracellular matrix formation and cell apoptosis via the Smad3 pathway.NEW & NOTEWORTHY In this work, we mainly discuss long noncoding RNA growth arrest-specific 5 (GAS5), acting in a renoprotective role via the Smad3/miRNA-142-5p axis, that modulates extracellular matrix formation and cell apoptosis. Overexpression of GAS5 effectively blocked renal fibrosis in vitro. This study reveals that GAS5 may represent as a novel and precision therapeutic target for alleviating renal fibrosis.

Artesunate relieves acute kidney injury through inhibiting macrophagic Mincle-mediated necroptosis and inflammation to tubular epithelial cell.

Artesunate is a widely used derivative of artemisinin for malaria. Recent researches have shown that artesunate has a significant anti-inflammatory effect on many diseases. However, its effect on acute kidney injury with a significant inflammatory response is not clear. In this study, we established a cisplatin-induced AKI mouse model and a co-culture system of BMDM and tubular epithelial cells (mTEC) to verify the renoprotective and anti-inflammatory effects of artesunate on AKI, and explored the underlying mechanism. We found that artesunate strongly down-regulated the serum creatinine and BUN levels in AKI mice, reduced the necroptosis of tubular cells and down-regulated the expression of the tubular injury molecule Tim-1. On the other hand, artesunate strongly inhibited the mRNA expression of inflammatory cytokines (IL-1ß, IL-6 and TNF-α), protein levels of inflammatory signals (iNOS and NF-κB) and necroptosis signals (RIPK1, RIPK3 and MLKL) in kidney of AKI mouse. Notably, the co-culture system proved that Mincle in macrophage can aggravate the inflammation and necroptosis of mTEC induced by LPS, and artesunate suppressed the expression of Mincle in macrophage of kidney in AKI mouse. Overexpression of Mincle in BMDM restored the damage and necroptosis inhibited by artesunate in mTEC, indicating Mincle in macrophage is the target of artesunate to protect tubule cells in AKI. Our findings demonstrated that artesunate can significantly improve renal function in AKI, which may be related to the inhibition of Mincle-mediated macrophage inflammation, thereby reducing the damage and necroptosis to tubular cells that provide new option for the treatment of AKI.

Renoprotective Effect of Oridonin in a Mouse Model of Acute Kidney Injury via Suppression of Macrophage Involved Inflammation.

Ischemia-reperfusion injury (IRI) is the major cause of acute kidney injury (AKI). The previous studies demonstrated that Oridonin can protect kidney against IRI-induced AKI, but the underlying molecular mechanism is unclear. In this study, it showed that Oridonin significantly improved kidney damage, and inhibited the expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and MCP-1, as well as macrophage marker F4/80 in kidney and the secretion of inflammatory cytokins in serum of AKI mice in vivo. In addition, Oridonin also effectively reduced the expression and secretion of lipopolysaccharide (LPS)-induced inflammatory factors in macrophage cell line RAW264.7 in vitro. Notably, Oridonin strongly downregulated Mincle and AKT/nuclear factor-kappaB (NF-κB) signaling both in vivo and in vitro, and the results of cellular recovery experiments of overexpression of Mincle in macrophage suggested that Oridonin suppressed inflammatory response of macrophage through inhibiting Mincle, which may be the underlying mechanism of Oridonin improving injury in kidney of AKI mice. In summary, the above results indicated that Oridonin can protect kidney from IRI-induced inflammation and injury by inhibiting the expression of Mincle in macrophage.

Smad3 deficiency promotes beta cell proliferation and function in db/db mice via restoring Pax6 expression.

Rationale: Transforming Growth Factor-beta (TGF-ß) /Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases, but its role in beta cell function and type 2 diabetes is unknown. Methods: The role of Smad3 in beta cell function under type 2 diabetes condition was investigated by genetically deleting Smad3 from db/db mice. Phenotypic changes of pancreatic islets and beta cell function were compared between Smad3 knockout db/db (Smad3KO-db/db) mice and Smad3 wild-type db/db (Smad3WT-db/db) mice, and other littermate controls. Islet-specific RNA-sequencing was performed to identify Smad3-dependent differentially expressed genes associated with type 2 diabetes. In vitro beta cell proliferation assay and insulin secretion assay were carried out to validate the mechanism by which Smad3 regulates beta cell proliferation and function. Results: The results showed that Smad3 deficiency completely protected against diabetes-associated beta cell loss and dysfunction in db/db mice. By islet-specific RNA-sequencing, we identified 8160 Smad3-dependent differentially expressed genes associated with type 2 diabetes, where Smad3 deficiency markedly prevented the down-regulation of those genes. Mechanistically, Smad3 deficiency preserved the expression of beta cell development mediator Pax6 in islet, thereby enhancing beta cell proliferation and function in db/db mice in vivo and in Min6 cells in vitro. Conclusions: Taken together, we discovered a pathogenic role of Smad3 in beta cell loss and dysfunction via targeting the protective Pax6. Thus, Smad3 may represent as a novel therapeutic target for type 2 diabetes prevention and treatment.

Identification of Hub Genes Associated with the Development of Acute Kidney Injury by Weighted Gene Co-Expression Network Analysis.

BACKGROUND: Acute kidney injury (AKI) is a severe clinical syndrome, causing a profound medical and socioeconomic burden worldwide. This study aimed to explore underlying molecular targets related to the progression of AKI. METHODS: A public database originated from the NCBI GEO database (serial number: GSE121190) and a well-established and unbiased method of weighted gene co-expression network analysis (WGCNA) to identify hub genes and potential pathways were used. Furthermore, the unbiased hub genes were validated in 2 classic models of AKI in a rodent model: chemically established AKI by cisplatin- and ischemia reperfusion-induced AKI. RESULTS: A total of 17 modules were finally obtained by the unbiased method of WGCNA, where the genes in turquoise module displayed strong correlation with the development of AKI. In addition, the results of gene ontology revealed that the genes in turquoise module were involved in renal injury and renal fibrosis. Thus, the hub genes were further validated by experimental methods and primarily obtained Rplp1 and Lgals1 as key candidate genes related to the progression of AKI by the advantage of quantitative PCR, Western blotting, and in situ tissue fluorescence. Importantly, the expression of Rplp1 and Lgals1 at the protein level showed positive correlation with renal function, including serum Cr and BUN. CONCLUSIONS: By the advantage of unbiased bioinformatic method and consequent experimental verification, this study lays the foundation basis for the pathogenesis and therapeutic agent development of AKI.

Nodakenin alleviates renal ischaemia-reperfusion injury via inhibiting reactive oxygen species-induced NLRP3 inflammasome activation.

BACKGROUND: Acute kidney injury (AKI) is a vital contributor to chronic kidney disease and limited therapeutic options are existed to preserve the renal injury. The research presented here investigated the protective effect of nodakenin against AKI and the underlying mechanism. METHODS: The effect of nodakenin was investigated in ischaemia reperfusion-induced renal injury (IRI) of AKI mice and hypoxia-treated primary renal tubular cells. Briefly, renal functions including creatinine and urea nitrogen were determined and mechanisms associated inflammation were investigated by the advantage of immunohistochemistry, western blot, RT-PCR and flow cytometry. RESULTS: Deterioration of renal functions including and creatinine, urea nitrogen and tubular necrosis were observed in IRI-AKI model. In contrast, nodakenin strikingly alleviated the deterioration of creatinine, urea nitrogen and tubular necrosis when compared with IRI model. Moreover, nodakenin could significantly inhibit the expression of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6 and tumour necrosis factor-α both in hypoxia-treated primary renal tubular cells and in AKI model. Mechanistic studies revealed that nodakenin dramatically suppressed the production of reactive oxygen species and subsequent NLPR3 inflammasome activation. CONCLUSION: In summary, these findings provided a solid evidence base and a new drug option for the treatment of AKI.

Protocatechualdehyde attenuates obstructive nephropathy through inhibiting lncRNA9884 induced inflammation.

Persistent chronic inflammation and fibrosis product accumulation aggravate tubulointerstitial fibrosis (TIF), leading to the progression of chronic kidney disease. The aim of this study was designed to investigate the effect of protocatechualdehyde (PCA), a natural phenolic acid compound isolated from Salvia miltiorrhiza, on the unilateral ureteral obstruction (UUO)-induced fibrosis and inflammation and to elucidate the underlying mechanism in primary renal tubular epithelial cells (TECs). Results from the histology suggested that the moderate to severe deteriorations of renal dysfunction and the pathological changes in UUO could be relieved by PCA treatment. Mechanistic studies revealed that the effect of PCA was associated with the downregulation of Smad3 and NF-κB driven fibrosis and inflammation respectively. It is worth noting that PCA could inhibit the aberrant expression of inflammation cytokines such as iNOS, MCP-1, TNF-α in UUO, and IL-1ß-treated TECs. In addition, PCA also suppressed the expression of Smad3-dependent long noncoding RNA (lncRNA), 9884. Importantly, when overexpressing of lncRNA9884 in TECs by transfection of pcDNA3.1-lncRNA9884 plasmid, it revealed significant reversal of protein expression levels as that observed with only PCA, suggesting that PCA inhibits inflammation by mediating lncRNA9884/MCP-1 signaling pathway. Collectively, the current study establishes a foundational basis for PCA in future treatment of obstructive nephropathy.

Decursin inhibits the growth of HeLa cervical cancer cells through PI3K/Akt signaling.

Decursin, a coumarin compound isolated from Angelica gigas has been shown to possess multiple anti-tumor activities. But it's still little known about the effects associated with cervical cancer. To explore the anti-tumor role of decursin and gain insights into its underlying mechanisms, we analyzed proliferation in parallel with apoptosis and migration in HeLa cells. Our findings implied that decursin can provoke apoptosis, and inhibit cell proliferation, migration in HeLa cells. More importantly, decursin also inhibited the tumor growth in vivo. The mechanisms may be associated with the regulation of Akt activation, with implications for novel therapeutic strategies on cervical cancer.[Formula: see text].

BAY61-3606 protects kidney from acute ischemia/reperfusion injury through inhibiting spleen tyrosine kinase and suppressing inflammatory macrophage response.

Acute kidney injury (AKI) is a highly prevalent clinical syndrome with high mortality and morbidity. Previous studies indicated that inflammation promotes tubular damage and plays a key role in AKI progress. Spleen tyrosine kinase (Syk) has been linked to macrophage-related inflammation in AKI. Up to date, however, no Syk-targeted therapy for AKI has been reported. In this study, we employed both cell model of LPS-induced bone marrow-derived macrophage (BMDM) and mouse model of ischemia/reperfusion injury (IRI)-induced AKI to evaluate the effects of a Syk inhibitor, BAY61-3606 (BAY), on macrophage inflammation in vitro and protection of kidney from AKI in vivo. The expression and secretion of inflammatory cytokines, both in vitro and in vivo, were significantly inhibited even back to normal levels by BAY. The upregulated serum creatinine and blood urea nitrogen levels in the AKI mice were significantly reduced after administration of BAY, implicating a protective effect of BAY on kidneys against IRI. Further analyses from Western blot, immunofluorescence staining and flow cytometry revealed that BAY inhibited the Mincle/Syk/NF-κB signaling circuit and reduced the inflammatory response. BAY also inhibited the reactive oxygen species (ROS), which further decreased the formation of inflammasome and suppressed the mature of IL-1ß and IL-18. Notably, these inhibitory effects of BAY on inflammation and inflammasome in BMDM were significantly reversed by Mincle ligand, trehalose-6,6-dibehenate. In summary, these findings provided compelling evidence that BAY may be an efficient inhibitor of the Mincle/Syk/NF-κB signaling circuit and ROS-induced inflammasome, which may help to develop Syk-inhibitors as novel therapeutic agents for AKI.