Synthesis, antitumor activity evaluation of 2-selenocyano-3-selenocyanoalkyloxyestradiols with a bisselenocyanate structure.

The combination of steroid structure and selenocyano group offers high potential for the design and synthesis of new potential anti-tumor drugs. Beginning with estradiol, a series of 2-selenocyano-3-selenocyanoalkyloxyestradiol derivatives with remarkable antiproliferative activity was synthesized. Additionally, a 2,4-bisselenocyanoestradiol was synthesized by directly selenocyanating estradiol diacetate. It was found that the cytotoxicity of 2-selenocyano-3-selenocyanoalkyloxyestradiol derivatives was significantly increased in comparison to the corresponding monoselenocyanate precursor, whereas the cytotoxicity of the 2, 4-bisselenocyanoestradiol derivative was significantly reduced compared to the respective monosubstituted precursor. The introduction of the second selenocyano group at different locations of estradiol shows a various impact on the cytotoxicity of the compounds. Among them, compound 3e showed the best cytotoxicity, with an IC50 value of less than 5 µM against the tested tumor cells, and strong inhibitory activities against HeLa and MCF-7 cell xenograft tumors in zebrafish, suppressing tumor cell migration and neovascularization. Notably, compound 3e was more effective at inhibiting neovascularization of MCF-7 cell xenograft tumors than the positive control 2-methoxyestradiol. Furthermore, compound 3e showed excellent anti-oxidative stress effect in zebrafish. Therefore, these estrogen bisselenocyanate compounds may be promising anti-tumor agents, warranting further investigation.

Hirudin ameliorates diabetic nephropathy by inhibiting Gsdmd-mediated pyroptosis.

Our group previously reported that hirudin ameliorated diabetic nephropathy (DN) in streptozotocin (STZ)-injected rats, but the mechanism remained largely unknown. Therefore, we further explored its possible mechanism. We subcutaneously injected 5 U hirudin into STZ-induced WT mice or Gasdermin D (Gsdmd)-/- (KO) mice daily for 12 weeks, respectively, and evaluated their kidney injury. Next, glomerular endothelial cells (GECs), renal tubular epithelial cells (RTECs), and bone-marrow-derived macrophages (BMDMs) were isolated from WT mice and treated with hirudin in the presence of high glucose/lipopolysaccharides and ATP to measure the release of interleukin-18 and interleukin-1ß. Kidney injury induced by STZ injection was significantly ameliorated by hirudin through inhibiting Gsdmd-mediated pyroptosis in the mice, not Caspase 1-mediated apoptosis. Meanwhile, hirudin also suppressed pyroptosis in primary GECs, RTECs, and BMDMs in vitro. Moreover, the deletion of Gsdmd reduced pyroptosis and kidney injury both in vivo and in vitro. We also found that hirudin regulated the expression of Gsdmd by inhibiting interferon regulatory factor 2 (Irf2). Hirudin ameliorated Gsdmd-mediated pyroptosis by inhibiting irf2, leading to the improvement of kidney injury. Therefore, hirudin might serve as a potential therapeutic strategy to treat DN.

CMKLR1 Antagonist Alpha-NETA Protects against Diabetic Nephropathy in Mice.

INTRODUCTION: Diabetic nephropathy (DN) is a common complication in diabetic patients. Chemerin, a novel adipokine, has been associated with renal damage in DN. The chemerin chemokine-like receptor 1 (CMKLR1) has been reported to participate in DN. In this study, we aimed to investigate the effect of a CMKLR1 antagonist, 2-(anaphthoyl)ethyltrimethylammonium iodide (α-NETA), on DN. METHODS: To induce diabetes, 8-week-old male C57BL/6J mice were given a single intraperitoneal injection of 65 mg/kg streptozotocin (STZ). Diabetic mice were randomly assigned to receive daily doses of 0, 5, or 10 mg/kg α-NETA for 4 weeks. RESULTS: α-NETA dose-dependently induced body weight and reduced fasting blood glucose levels in STZ-induced diabetic mice. Furthermore, α-NETA significantly reduced the expressions of renal injury markers, including serum creatinine, kidney weight/body weight, urine volume, total proteins, and albumin in the urine, and increased creatinine clearance. Periodic acid-Schiff staining also indicated that α-NETA could effectively ameliorate renal injuries in DN mice. In addition, α-NETA inhibited renal inflammation and the expressions of chemerin and CMKLR1 in mice with DN. CONCLUSION: In summary, our findings suggested that α-NETA has beneficial effects on the management of DN. Specifically, α-NETA effectively ameliorated renal damage and inflammation in a dose-dependent manner in mice with DN. Thus, targeting the chemerin and CMKLR1 axis with α-NETA may be a promising therapeutic strategy for the treatment of DN.

Synthesis of estrone selenocyanate Compounds, anti-tumor activity evaluation and Structure-activity relationship analysis.

Introducing different functional groups into steroid can bring unexpected changes in biological activity of the steroid. Using estrone as a raw material, through the functional group conversion and modification of the 17-carbonyl, the structural fragments with selenocyano groups were instilled in the form of amide, ester, and oxime ester, respectively, and various 17-substituted estrone selenocyanate derivatives were synthesized. In addition, different 3-substituted estrone selenocyanate derivatives were synthesized by introducing different selenocyanoalkoxy fragments into the 3-position of estrone in the form of alkyl ether. Furthermore, the selenocyano-containing moieties were embedded into the 2-position of estrone by means of amide, affording diverse 2-selenocyanoamide-estrone derivatives. The antiproliferative activities of the target compounds were screened by selecting tumor cell lines related to the expression of human hormones. The results showed that the introduction of selenocyano group into estrone could endow estrone with significant biological activity of inhibiting the proliferation of tumor cells. Structure-activity relationship research showed that the cytotoxicity of 3-selenocyanoalkoxy-estrone was further increased with the extension of alkyl carbon-chain within 8 carbon chain lengths. In addition, the cytotoxicity of the products with selenocyano via the form of amide was stronger than that of ester or ether. Selenocyano moiety instilled at the 2-position of estrone in the form of amide was more cytotoxic than that of 17- or 3-position. Among them, compound 21a has better inhibitory activity on tested tumor cells than positive controls Abiraterone and 2-methoxyestradiol. Research showed that the compound 21c induced programmed apoptosis in Sk-Ov-3 cancer cells, and compound 17d inhibited significantly the growth of human cervical cancer zebrafish xenografts in vivo, offering useful insights into the synthesis of steroid antitumor drugs.

LncRNA TTN-AS1/miR-134-5p/PAK3 axis regulates the radiosensitivity of human large intestine cancer cells through the P21 pathway and AKT/GSK-3ß/ß-catenin pathway.

Radiotherapy is an important adjuvant treatment for large intestine cancer even though it does not cause any response in many patients. The present study aimed to investigate the effects of the TTN antisense RNA 1 (TTN-AS1) long noncoding RNA (lncRNA) on radiotherapy dynamics of large intestine cancer cells and to explore the underlying molecular mechanisms. TTN-AS1 expression was evaluated by reverse-transcription quantitative polymerase chain reaction, western blot, and cellular immunofluorescence, and flow cytometry analysis was used to measure apoptosis. Radiotherapy was simulated in vitro by exposing cancer cells to X-ray. TTN-AS1 was highly expressed in large intestine cancer cells after an X-ray exposition for 24 hr. TTN-AS1 knockdown improved the radiosensitivity of large intestine cancer cells and promoted apoptosis by increasing Bax/Bcl2 protein expression and the active-caspase 3/caspase 3 ratios following X-ray treatment. In addition, TTN-AS1 negatively regulated miR-134-5p expression, and miR-134-5p-mimic transfection decreased PAK3 protein expression in large intestine cancer cells. Importantly, TTN-AS1 promoted PAK3 and P21 protein expression in HT29 cells after X-ray treatment. Moreover, the knockdown of P21 protein expression improved radiosensitivity and promoted X-ray-induced apoptosis of HT29 cells. Finally, PAK3 knockdown expression decreased the p-AKT/AKT and p-GSK-3ß/GSK-3ß ratios and promoted the ß-catenin transfer from the nucleus to the cytoplasm. These data suggest that the TTN-AS1 lncRNA promoted resistance to radiotherapy of large intestine cancer cells by increasing PAK3 expression via miR-134-5p inhibition, and this may be related to the P21 and AKT/GSK-3ß/ß-catenin pathway.

Hirudin Reduces the Expression of Markers of the Extracellular Matrix in Renal Tubular Epithelial Cells in a Rat Model of Diabetic Kidney Disease Through the Hypoxia-Inducible Factor-1α (HIF-1α)/Vascular Endothelial Growth Factor (VEGF) Signaling Pathway.

BACKGROUND This study aimed to investigate the effects of hirudin on the production of extracellular matrix (ECM) factors by renal tubular epithelial cells in a rat model of diabetic kidney disease (DKD) and HK-2 human renal tubule epithelial cells. MATERIAL AND METHODS Sprague-Dawley rats were divided into the normal control group (n=10), the normal control+hirudin group (n=10), the DKD model group (n=12) and the DKD+hirudin group (n=12). At the end of the study, renal histopathology was undertaken, and the expression of type IV collagen, fibronectin, hypoxia-inducible factor-1alpha (HIF-1alpha), and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). HK-2 cells were cultured in glucose and treated with hirudin. Protein and mRNA expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF were evaluated following knockdown or overexpression of HIF-1alpha. RESULTS Hirudin significantly improved renal function in the rat model of DKD (P<0.01), and significantly down-regulated the expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF proteins (P<0.05). The expression of ECM associated proteins was increased in HK-2 cells treated with high glucose and reduced in the high glucose+shRNA HIF-1alpha group (P<0.05). Compared with the control group, the expression of ECM associated proteins was increased in the HIF-1alpha over-expressed group, and decreased following treatment with hirudin (P<0.05). CONCLUSIONS Hirudin reduced the expression of markers of ECM by inhibiting the HIF-1alpha/VEGF signaling pathway in DKD renal tubular epithelial cells.

Synthesis and Antiproliferative Activity Evaluation of B-norcholesterol-6- amide Compounds.

BACKGROUND: The structure modification of steroids is commonly used to change the biological activity of steroids in medicinal chemistry. In recent years, it has been found that some derivatives derived from the structural modification of cholesterol display good inhibitory activity against tumor cell proliferation in vitro. METHODS: Using cholesterol as the starting material, different types of B-norcholesterol-6-amide derivatives were synthesized by the reaction of 6-carboxyl-B-norcholesterol with different alkyl amines or 6-amino-B-norcholesterol with different acyl chlorides. The inhibitory activity of compounds on the proliferation of tumor cell lines was investigated by the MTT method. RESULTS: The results showed that the B-norcholesterol-6-amide compounds displayed distinct cytotoxicity against Sk-Ov-3 cells but caused no obvious damage against HEK-293T cells. Additionally, the steroidal amide derivatives formed from 6-amino-B-norcholesterol showed stronger cytotoxicity than those produced from 6-carboxyl-B-norcholesterol. Specially, compounds with chloroalkyl structure displayed significant inhibitory activity against all tumor cells tested. Among them, compounds 19-21 showed cytotoxicity like 2-methoxyestradiol as a positive control, and the IC₅₀ value of compound 20 on HeLa cells was 3.9 µM. CONCLUSION: After introducing chloroalkyl acyl groups into 6-position of 6-amino-B-norcholesterol, the cytotoxicity of resulting B-norcholesterol-6-amide compounds can be greatly enhanced.

Discovery and biological evaluation of pregnenolone selenocyanoamides with potential anticancer and antimicrobial activities.

Starting with pregnenolone, a 20-carbonyl group was converted into an amino group through a series of chemical reactions. This amino group was further converted into selenocyanoalkylamide, leading to the synthesis of six pregnenolone selenocyanoalkylamide derivatives. These compounds were then screened for antitumor activity in vitro, yielding promising results. Compounds 4b-4f show higher inhibitory activity than the positive control abiraterone and 2-methoxyestradiol, with IC50 values lower than 10 µmol/L against breast, ovarian, and cervical cancer cell lines that closely related to human hormone expression levels. The Annexin V assay of compound 4f revealed that compounds inhibited tumor cell proliferation primarily through the induction of programmed apoptosis. The zebrafish test results indicated that compound 4d had significant inhibitory activity against MCF-7 cell xenografts in vivo. Moreover, the antibacterial test indicated that compounds 4a and 4d-4e had better inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) than the positive controls vancomycin and ampicillin. These results suggest that these compounds may hold promise as novel antitumor agents or antimicrobial agents for further study.

Synthesis, characterization and application of emamectin-alkaline lignin conjugate with photolysis resistance and systemic translocation.

Controlled release formulations (CRFs) are a key technical approach for the sustainable development of pesticides. In this study, a CRF conjugate (emamectin-alkaline lignin, EB-AL) was successfully prepared using alkaline lignin as the substrate, with amide bond connecting emamectin and alkaline lignin. The structure and morphology of the conjugate were characterized using IR, 1HNMR, elemental analysis, SEM and TG. The release of EB-AL showed that the conjugate maintained its original structure when released in 50 % methanol-water and soil column, and the amide bond remained intact. The anti-photolysis test revealed that EB-AL had a 3.5 times higher photolysis half-life T0.5 than the general emamectin suspension concentrate (EB-SC). Bioactivity tests in the greenhouse demonstrated that EB-AL possessed a longer insecticidal duration and good biosafety. Ostrinia nubilalis lethality rate remained above 70 % for 19 days, while EB-EC, the control, had a rate of <50 % after 11 days of application. Additionally, EB-AL conjugate demonstrated excellent systemic translocation in plants, likely due to its ability to mediate alkaline lignin.

Straightforward synthesis of steroidal selenocyanates through oxidative umpolung selenocyanation of steroids and their antitumor activity.

Straightforward access to steroidal selenocyanates in a single assembly step from steroids remains a significant challenge. However, the development of novel method for the synthesis of steroidal selenocyanates and further investigation of their bioactivities have largely lagged behind. In this work, selenocyano groups were directly introduced into the 17- or 21-position of pregnenolone, the 2-position of estradiol, and the 16-position of estrone. A total of 16 estrogen selenocyanate derivatives with diverse structures were synthesized, and the tumor cell lines closely related to the expression level of estrogen were used to investigate the inhibitory activity of the target products on tumor cell proliferation in vitro. The results revealed that the 17-selenocyano-substituted pregnenolone selenocyanate derivatives 1b-3b exhibit obvious inhibitory activity against the tested tumor cell lines. Additionally, the 2-selenocyano-substituted estradiol derivatives and 16-selenocyano-substituted estrone derivatives exhibit selective inhibitory on HeLa cell lines. Among them, 2-selenocyano-3-methoxyestradiol-17-benzoate (7e) displayed an IC50 value of 4.1 µM against HeLa cells and induced programmed apoptosis in HeLa cancer cells. Furthermore, compound 7e could significantly inhibit the growth of human cervical cancer xenografts in zebrafish in vivo. This approach provides new insights for future steroid antitumor drug design.

Synthesis and antitumor activity of some cholesterol-based selenocyanate compounds.

The introduction of selenium-containing functional groups into steroids to study the biological activities of related derivatives is rarely reported in the literature. In the present study, using cholesterol as raw material, four cholesterol-3-selenocyanoates and eight B-norcholesterol selenocyanate derivatives were synthesized, respectively. The structures of the compounds were characterized by NMR and MS. The results of the in vitro antiproliferative activity test showed that the cholesterol-3-selenocyanoate derivatives did not exhibit obvious inhibitory on the tested tumor cell lines. However, the B-norcholesterol selenocyanate derivatives obtained by structural modification of cholesterol showed good inhibitory activity against the proliferation of tumor cell. Among them, compounds 9b-c, 9f and 12 showed similar inhibitory activity against tested tumor cells as positive control 2-methoxyestradiol, and better than Abiraterone. At the same time, these B-norcholesterol selenocyanate derivatives displayed a strong selective inhibitory against Sk-Ov-3 cell line. Except for compound 9g, the IC50 value of all B-norcholesterol selenocyanate compounds against Sk-Ov-3 cells was less than 10 µM, and compound 9d was 3.4 µM. In addition, Annexin V-FITC/PI double staining was used to analyze the cell death mechanism. The results showed that compound 9c could induce Sk-Ov-3 cells to enter programmed apoptosis in a dose-dependent manner. Furthermore, the in vivo antitumor experiments of compound 9f against zebrafish xenograft tumor showed that 9f displayed obvious inhibitory effect on the growth of human cervical cancer (HeLa) xenograft tumor in zebrafish. Our results provide new thinking for the study of such compounds as new antitumor drugs.

Glycyrrhizic acid exhibits strong anticancer activity in colorectal cancer cells via SIRT3 inhibition.

Sirtuin-3 (SIRT3) has been described as a colorectal cancer oncogene and to be regulated by glycyrrhizic acid (GA). However, few studies have explored the interaction between GA and SIRT3. Therefore, in the present study, we showed that GA could significantly decrease SIRT3 protein levels in SW620 and HT29 cells in a dose-dependent manner. Then, we overexpressed SIRT3 by lentivirus infection on SW620 and HT29 cells. We found that, in vitro, GA treatment significantly decreased cell viability, cell clone number, and invasion and migration number, besides significantly increasing apoptosis. Also, GA treatment significantly decreased the Bax/Bcl2 protein ratio and the expression of Cyclin D1, CDK2, CDK4, MMP-9, N-cadherin, and vimentin in SW620 and HT29 cells. Meanwhile, the SIRT3 overexpression could significantly reverse these changes. Moreover, the GA treatment could significantly decrease the weight of xenograft tumor tissues and its SIRT3 protein levels in vivo, while SIRT3 overexpression reversed these effects. Overall, GA inhibited the proliferation, invasion, and migration of colorectal cancer cells, and induced their apoptosis by SIRT3 inhibition.

Synthesis and antiproliferative evaluation of some novel estradiol selenocyanates.

Selenocyano fragments with different structural characteristics have been successfully installed into the 3- and 17-position of estradiol through the etherification and esterification of its 3 or 17-hydroxyl group respectively. A total of 12 new estradiol selenocyanates were synthesized and their structures were characterized by NMR and HRMS. The tumor cell lines related to the expression of human hormones were selected as the screening objects, and the antiproliferative activity of the target compounds was further investigated. The results showed that the introduction of selenocyano group in estradiol could endue estradiol with the activity of inhibiting tumor cell proliferation, and 3-selenocyanoalkyl estradiol ethers had stronger cytotoxicity than their 17-selenocyanocarboxylates counterpart. Among them, IC50 value of compound 3e on HeLa cells was 5.69 µM. The information obtained from the studies may be useful for the design and development of novel chemotherapeutic drugs.

Preparation and Insecticidal Activity Evaluation of Emamectin-Lignin Sulfonic Acid Conjugate with Antiphotolysis Property.

Controlled release formulations (CRFs) are considered an effective way to solve the low bioavailability of traditional pesticides. However, CRFs prepared by coating or encapsulation has the disadvantage of explosive release of the ingredients. Sustained-release pesticides prepared by coupling with a carrier can overcome this shortcoming. In the present study, an emamectin-lignin sulfonic acid conjugate (EB-SL), in which emamectin was connected via sulfonamide bonds with lignin, was prepared using sodium lignosulfonate as the carrier. The structure of the conjugate was characterized by IR, 1HNMR, and elemental analysis. The sustained-release results showed that EB-SL maintained its original structure when released in pure water and soil columns, and the sulfamide bond did not break. The photolysis test displayed that the photolysis half-life T 0.5 of EB-SL was increased by 1.5 times compared with the emamectin suspending concentrate (EB-SC). Bioactivity tests in the greenhouse showed that EB-SL not only had similar insecticidal toxicity to emamectin emulsion concentrate (EB-EC) against Ostrinia nubilalis but also displayed a longer duration. The lethality of EB-SL on O. nubilalis was maintained at more than 70% across 19 days, whereas EB-EC as the control was less than 50% after 11 days of application.

Ginkgo Biloba Extract EGB761 Ameliorates the Extracellular Matrix Accumulation and Mesenchymal Transformation of Renal Tubules in Diabetic Kidney Disease by Inhibiting Endoplasmic Reticulum Stress.

The study is aimed at investigating the effects of Ginkgo biloba extract EGB761 on renal tubular damage and endoplasmic reticulum stress (ERS) in diabetic kidney disease (DKD). A total of 50 C57BL/6 N mice were randomly divided into the normal group, DKD group, DKD+EGB761 group (36 mg/kg), and DKD+4-phenylbutyrate (4-PBA) group (1 g/kg). The DKD model was replicated by high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). Renal tubular epithelial cells (HK-2) were divided into the control group, high-glucose group (30 mmol/L), EGB761 group (40 mg/L, 20 mg/L, 10 mg/L), TM group, and TM+4-PBA group. After 8 weeks of administration, expressions of serum creatinine (Scr), blood urea nitrogen (BUN), 24 h urinary protein (24 h Pro), fasting blood glucose (FBG), ß 2-microglobulin (ß 2-MG), and retinol binding protein 4 (RBP4) of mice were tested. The pathological changes of renal tissue were observed. The expressions of extracellular matrix (ECM) accumulation and epithelial-mesenchymal transition (EMT) markers α-smooth muscle actin (α-SMA), E-cadherin, fibronectin, and collagen IV, as well as the ERS markers GRP78 and ATF6, were tested by Western blot, qPCR, immunohistochemistry, or immunofluorescence. EGB761 could decrease the Scr, BUN, 24 h Pro, and FBG levels in the DKD group, alleviate renal pathological injury, decrease urine ß 2-MG, RBP4 levels, and decrease the expression of α-SMA, collagen IV, fibronectin, and GRP78, as well as ATF6, while increase the expression of E-cadherin. These findings demonstrate that EGB761 can improve renal function, reduce tubular injury, and ameliorate ECM accumulation and EMT in DKD kidney tubules, and the mechanism may be related to the inhibition of ERS.

Tongluo Digui decoction treats renal injury in diabetic rats by promoting autophagy of podocytes.

OBJECTIVE: To investigate the effects of Tongluo Digui decoction on renal injury and streptozotocin-induced podocyte autophagy in diabetic rats. METHODS: Male Sprague-Dawley rats were randomly divided into six groups: normal, model, Tongluo Digui decoction (high, medium, and low dose) and valsartan. Streptozotocin was injected intraperitoneally to replicate the diabetic animal model. After 8 weeks, proteinuria was evaluated to establish the diabetic nephropathy model. Treatments were administered daily via the intragastric route. At 16 weeks after gavage, we determined 24 h urine protein concentration, and blood glucose, serum creatinine, and urea nitrogen concentrations. Then, rats were sacrificed, and kidneys were harvested and stained with periodic acid-Schiff to evaluate the pathological changes in glomeruli, including glomerular podocytes by transmission electron microscopy. Western blot analysis was used to determine the expression of nephrin, podocin, p62, beclin-1, LC3Ⅱ/Ⅰ, and p-mTOR/mTOR protein in kidney tissues. RESULTS: Compared with the model group, Tongluo Digui decoction was associated with decreases in 24 h urine protein concentration, and blood glucose, hemoglobin A1c, serum creatinine, urea nitrogen concentrations, total serum protein and albumin. Concurrently, mesangial mesenteric broadening and fusion of foot processes were reduced, the glomerular basement membrane was not significantly thickened, and the number of podocytes and the number of autophagosomes in the podocytes was increased. Further, expression of nephrin, podocin, LC3Ⅱ, and beclin-1 protein in kidney tissue was up-regulated, while expression of p62 protein was down-regulated and mTOR phosphorylation was inhibited. CONCLUSION: Tongluo Digui decoction may inhibit the progression of diabetic nephropathy by inhibiting mTOR phosphorylation, thereby increasing autophagy to protect podocytes and reducing proteinuria.

Hirudin reduces nephropathy microangiopathy in STZ-induced diabetes rats by inhibiting endothelial cell migration and angiogenesis.

OBJECTIVE: Kidney is the most common location of microangiopathy in diabetic patients, and we designed this study to investigate the effects of hirudin on renal microangiopathy in STZ-induced diabetes rats and in vitro. METHODS: We established a diabetes model by intraperitoneal injection of STZ and administered hirudin daily by subcutaneous injection. HE staining was used to assess kidney pathological changes. Western blot and immunochemistry was used to detect the protein expression. Glomerular endothelial cells (GEC) in normal rats were assessed by cell scratch test for migration ability and tubule formation experiment for angiogenesis ability. RESULTS: Compared with DN rats without any treatment, the serum creatinine, serum Cys C, 24-hour urine protein of DN rats with hirudin treatment were significantly decrease, the kidney/body weight and glomerular area of DN rats with hirudin treatment were all significantly decrease, and also significant improvement in renal pathology revealed by HE staining in DN rats after treating with hirudin. Moreover, we also found that hirudin coun not only significantly increase the prothrombin time and aivated partial thromboplastin time in DN rats, but also significantly decrease the expression of VEGF and TM-1 protein in kidney tissues of DN rats. In vitro, we found that high glucose could promote the migration and angiogensis of GEC, and significantly increased the expression of VEGF and Ang protein, but significantly decreased the expression of THBS1 and Arg1 protein. More importantly was that hirudin could inhibit the migration and angiogensis of GEC, and reversed HG-induced the expression of VEGF, Ang, THBS1 and Arg1 protein in GEC. In addition, we also found that hirudin could not only decrease HG-enhanced the activity of RhoA in GEC, but also decrease HG-enhanced the expression of p-MYPT1/MYPT1, p-p38/p38 protein in GEC. CONCLUSION: Hirudin reduces nephropathy microangiopathy in STZ-induced diabetes, and might be related to hirudin inhibiting glomerular endothelial cell migration and angiogenesis through Rho-kinase and subsequent p38MAPK/NF-kB signaling pathway.

Hirudin Protects Against Kidney Damage in Streptozotocin-Induced Diabetic Nephropathy Rats by Inhibiting Inflammation via P38 MAPK/NF-κB Pathway.

BACKGROUND: Inflammation-induced podocyte apoptosis plays an important role in kidney injury during diabetic nephropathy (DN). Hirudin (HIR), a natural compound extracted from leeches, can inhibit inflammation. However, whether HIR can protect the kidneys against inflammation during DN is unknown. In the present study, we aimed to study the effects of HIR on kidney damage in a DN rat model and explore its anti-inflammatory properties. METHODS: A streptozotocin-induced DN rat model was generated, and HIR was administered subcutaneously. Immortal podocytes and primary peritoneal macrophages were used for vitro studies. Hematoxylin and eosin staining was used to evaluate renal pathological changes; quantitative polymerase chain reaction and immunoblotting were used to detect gene expression; and TUNEL staining was used to detect apoptotic cells. RESULTS: Our results showed that HIR protected against renal injury, as indicated by kidney weight/body weight, serum creatinine, renal pathological changes, blood urea nitrogen, and detection of urine proteins. Notably, HIR treatment reduced macrophage infiltration, pro-inflammatory cytokine expression, and podocyte apoptosis in the kidney tissues of DN rats. In vitro, high glucose (HG) induced the activation of M1 macrophages, which was accompanied by increased podocyte apoptosis. HIR could decrease HG-induced podocyte apoptosis and suppress pro-inflammatory cytokine expression in podocytes in vitro. This was achieved via inhibition of p38 MAPK/NF-κB activation in renal tissues and podocytes. CONCLUSION: HIR could inhibit inflammation via the p38 MAPK/NF-κB pathway, prevent podocyte apoptosis, and protect against kidney damage in a DN rat model.