Self-Priming DNA Polymerization-Propelled Stochastic Walkers on Magnetic Microbeads for Amplified Detection of miRNA.

Sensitive detection of miRNA targets in complex biological samples possesses great value in biopsy analysis and disease diagnosis but is still challenging because of low abundance and nonspecific interferences. In this work, self-primer DNA polymerization-propelled stochastic walkers (SWs) were proposed to detect miRNA-24 by combining magnetic microbeads (MMBs) and flow cytometry. The MMBs not only provide a three-dimensional interface for DNA walkers but also facilitate the enrichment and isolation of RNA targets from complex biological samples such as serum. The SWs can be initiated to walk through the entire surface of MMBs and transduce RNA walking into amplified fluorescence signals, with the detection limit of miRNA-24 at 0.95 pM. Moreover, this strategy integrating with flow cytometry was demonstrated to have good specificity with other homologous miRNAs. This platform offers promising applications in RNA biosensing and biomedical diagnostics.

Effect of low energy shock wave on testicular microenvironment homeostasis in rats.

OBJECTIVE: To further investigate whether two sets of low-energy extracorporeal shock waves (LESWs) impulse parameters, i.e., 0.02 mJ/mm2 for 500 impulses and 0.04 mJ/mm2 for 500 impulses, which have been shown to directly affect the testes, can promote testicular spermatogenesis or positively regulate homeostasis of the testicular microenvironment. METHODS: (1) Twenty-four experimental rats were randomly divided into a 0.02 mJ/mm2 500 impulses group (L1 group), a 0.04 mJ/mm2 500 impulses group (M1 group), a sham intervention group (S group) and a blank control group (N group). The experiment period was 8 weeks. (2) Apoptosis of the spermatogenic cells in the left testicle was detected by the TUNEL method, VEGF and eNOs protein expression was detected by immunohistochemistry, and histomorphological changes were observed in PAS-stained sections. Moreover, the morphologies of the spermatogenic tubules and testicular stroma were quantitatively analyzed by stereological analysis. The right testicle was used for Western blot detection of the protein expression levels of Bax, Cytochrome C, Caspase-3, Bcl-2, VEGF and eNOs. RESULTS: Compared with the other three groups, the rate of M1 testicular germ cell apoptosis induced by shock treatment was higher, the expression levels of proapoptotic proteins increased significantly while that of the antiapoptotic protein was lower, and the suppression of cell proliferation correlated with the protein expression levels. Additionally, with respect to the absolute volume of the seminiferous tubules, the absolute interstitial testicular volume notably increased, producing a series of biological effects working against testicular sperm production and function. However, there was no significant difference between the L1 group and the N and S groups. CONCLUSIONS: LESWs treatment with impulse parameters of 0.02 mJ/mm2 for 500 impulses showed a better protective effect on testicular spermatic function in rats and has a positive regulatory biological effect.

Comprehensive analysis of the importance of PLAUR in the progression and immune microenvironment of renal clear cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a common type of kidney cancer with a high mortality rate, and the discovery of new therapeutic markers is essential to improve patient survival. The plasminogen activator urokinase receptor (PLAUR) plays key roles in tissue remodeling and extracellular matrix degradation, which contribute to invasion and metastasis, a major feature of tumor malignancy. The role of PLAUR in ccRCC pathology has not been deeply studied. In this study, we collected the mRNA expression data of 33 tumor types, each derived from human patients obtained from TCGA database, and comprehensively analyzed the correlation between the expression of PLAUR in tumors and prognosis. Then, we studied the relationship between PLAUR expression in ccRCC and specific clinical features of ccRCC patients. In addition, we analyzed the function and mechanism of PLAUR in ccRCC. Our results showed that PLAUR was significantly overexpressed in ccRCC and that both PLAUR levels and PLAUR methylation levels significantly correlated with poor prognosis. Our results also suggest that PLAUR is involved in the progression of ccRCC. The results of functional and mechanistic analysis of PLAUR showed that PLAUR is involved in inflammatory and immune-related pathways in ccRCC; other data showed that PLAUR expression may affect the infiltration of multiple immune cell types in ccRCC and that PLAUR levels were significantly and positively correlated with the expression of immune checkpoints. In conclusion, our findings suggest that high PLAUR expression can promote the progression of ccRCC to poor prognosis, and thus PLAUR may serve as both a potential marker for predicting macrophage infiltration and immune microenvironment status and as an important immunotherapy target for ccRCC.

Four Highly Oxygenated Sesquiterpenoids from the Fruits of Illicium micranthum Dunn.

Four highly oxygenated sesquiterpenoids, illimicranolides A (1) and B (2), and illicinolides E (3) and F (4), were obtained from the fruits of Illicium micranthum Dunn, as well as one known analog, illicinolide B (5). The chemical structures of 1-4 were determined comprehensively by 1D (1 H and 13 C) and 2D (HMBC, HSQC, 1 H-1 H COSY, and ROESY) NMR, and HR-ESI-MS data. Structurally, compound 1 was an unprecedented sesquiterpenoid with a 5/5/6/5-fused tetracyclic ring system and was the first seco-prezizaane sesquiterpenoid featuring a 11,8-γ-lactone ring. Compounds 3 and 4 were the fifth and sixth examples of illicinolide-type sesquiterpenoids. Moreover, compound 1 demonstrated neurotrophic activity of NGF-induced PC12 cells with differentiation rate of 10.34 % at a concentration of 10 µM.

Auto-cycling primer extension for amplified microRNA detection.

The current polymerase-based nucleic acid amplification techniques cannot maintain continuous polymerization reactions unless by changing the temperature or adding additional reagents (e.g. the second enzyme or betaine), which undoubtedly increases the cost and operation steps. Herein, a new isothermal nucleic acid amplification technique, termed auto-cycling primer extension (APE), is presented. It repeatedly extends short DNA primers to longer DNA hairpins, by combining a DNA-based copy-and-release hairpin (CRH) and palindromic sequence design. The experimental results showed that we could realize the amplification detection of miRNA by a reasonable probe design.

Hypopurolides A - G, Labdane Diterpenoids from Hypoestes purpurea and Their Nitric Oxide Inhibitory Activity.

Seven new labdane diterpenoids, hypopurolides A-G (1-7) were discovered from the aerial part of Hypoestes purpurea, along with one known analog, hypopurin D (8). The structures of 1-7 were characterized based on 1 H-, 13 C-, and 2D-NMR, and HR-ESI-MS spectra. The absolute configurations of 1-7 were defined by single-crystal X-ray diffraction and electronic circular dichroism (ECD) data. Compounds 1-8 were tested for their nitric oxide (NO) inhibitory and cytotoxic effects. Compound 6 displayed moderate inhibitory effect toward LPS-induced NO release in RAW 264.7 cells with an IC50 value of 41.50 µM.

Silencing of LINC01963 enhances the chemosensitivity of prostate cancer cells to docetaxel by targeting the miR-216b-5p/TrkB axis.

Docetaxel (DTX) treatment effectively prolongs the overall survival of patients with prostate cancer. However, most patients eventually develop resistance to chemotherapy and experience tumor progression or even death. Long noncoding RNAs (lncRNAs) affect docetaxel chemosensitivity. However, the biological role and regulatory mechanisms of lncRNAs in docetaxel-resistant prostate cancer remain unclear. Differences in lncRNAs were evaluated by lncRNA sequencing and evaluated using quantitative real-time polymerase chain reaction, and TrkB expression was measured through western blot analysis. Proliferation was measured using the MTS, while apoptosis and cell cycle were measured using flow cytometry. In addition, migration and invasion were measured using transwell assays. Forty-eight female BALB/c nude mice were used for subcutaneous tumorigenicity and lung metastasis assays. We found that LINC01963 was overexpressed in the PC3-DR cells. LINC01963 silencing enhanced the chemosensitivity of PC3-DR to docetaxel and inhibited tumorigenicity and lung metastasis, while LINC01963 overexpression enhanced the chemoresistance of PC3 cells to docetaxel. It was found that LINC01963 bind to miR-216b-5p. The miR-216b-5p inhibitor reversed the suppressive effect of sh-LINC01963 on PC3-DR cell proliferation, migration, and invasion. Furthermore, miR-216b-5p can bind to the 3'-UTR of NTRK2 and inhibit TrkB protein levels. TrkB enhances docetaxel resistance in prostate cancer and reverses the effects of LINC01963 silencing and miR-216b-5p overexpression. In conclusion, silencing LINC01963 inhibited TrkB protein level to enhance the chemosensitivity of PC3-DR to docetaxel by means of competitively binding to miR-216b-5p. This study illustrates that LINC01963 is a novel therapeutic target for treating prostate cancer patients with DTX resistance.

A novel FRET-based dendritic hybridization chain reaction for tumour-related mRNA imaging.

A novel FRET-based dendritic hybridization chain reaction (D-HCR) for TK1 mRNA imaging in living cells was developed. Compared with traditional complex D-HCR methods, it includes the advantages of having a simple design, an accurate signal and is suitable for use with living cells.

CircSERPINA3 regulates SERPINA3-mediated apoptosis, autophagy and aerobic glycolysis of prostate cancer cells by competitively binding to MiR-653-5p and recruiting BUD13.

BACKGROUND: Prostate cancer (PCa) belongs to an epithelial malignancy that occurs in the prostate gland and is the most common malignancy of the male genitourinary system. Referring to related literature, circSERPINA3 has been reported to be up-regulated in PCa. However, its biological function remains unclear. PURPOSE: This study aimed to reveal the specific role and relevant molecular mechanism of circSERPINA3 in PCa. METHODS: RT-qPCR was used to examine gene expression and functional analyses were conducted to verify the effect of circSERPINA3 on cell apoptosis, autophagy and aerobic glycolysis in PCa cells. Mechanism assays were applied to evaluate the relationship among circSERPINA3/miR-653-5p/SERPINA3/BUD13. RESULTS: CircSERPINA3 was verified to be up-regulated in PCa cells and to inhibit cell apoptosis while promoting aerobic glycolysis and autophagy in PCa cells. CircSERPINA3 and SERPINA3 were also testified to bind to miR-653-5p through a line of mechanism experiments. Moreover, it was discovered that circSERPINA3 could stabilize SERPINA3 mRNA via recruiting BUD13. Additionally, SERPINA3 was verified to inhibit cell apoptosis, while promoting aerobic glycolysis and autophagy in PCa cells. CONCLUSIONS: Our study suggested that circSERPINA3 regulated apoptosis, autophagy and aerobic glycolysis of PCa cells by competitively binding to miR-653-5p and recruiting BUD13.

A MnO2 nanosheet-mediated photo-controlled DNAzyme for intracellular miRNA cleavage to suppress cell growth.

Certain miRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed, thus, representing a potential new class of targets for therapeutic intervention. RNA-cleaving DNAzymes, mainly aimed at mRNA, have shown potential as therapeutic agents for various diseases. However, it's rarely reported that a DNAzyme was used for intracellular miRNA cleavage to suppress cell growth. Herein, we have developed a MnO2 nanosheet-mediated photo-controlled DNAzyme (NPD) for intracellular miRNA cleavage to suppress cell growth. MnO2 nanosheets adsorb photocaged DNAzymes, protect them from enzymatic digestion, and efficiently deliver them into cells. In the presence of intracellular glutathione (GSH), MnO2 nanosheets are reduced to Mn2+ ions, which serve as cofactors of the 8-17 DNAzyme for miRNA cleavage. Once the DNAzyme is activated by light, it can cyclically cleave endogenous miR-21 inside cells, which would suppress cancer cell migration and invasion, and finally induce cancer cell apoptosis.

A randomized, active-controlled, multicentre clinical trial to evaluate the efficacy and safety of oral sitafloxacin versus levofloxacin in Chinese adults with acute uncomplicated or complicated urinary tract infection.

PURPOSE: To evaluate the efficacy and safety of oral sitafloxacin versus levofloxacin in Chinese adults with acute uncomplicated urinary tract infection (UTI) or complicated UTI. METHODS: In this randomized, active-controlled clinical trial, the patients with acute uncomplicated UTI were randomized to receive sitafloxacin 100-mg once-daily (qd) or levofloxacin 500-mg qd orally for 3-5 days. The patients with complicated UTI were randomized to receive sitafloxacin 100-mg twice daily or levofloxacin 500-mg qd orally for 10-14 days. The primary endpoint was the clinical efficacy at test-of-cure (TOC) visit. RESULTS: At TOC visit, the clinical cure rate was 89.2% (58/65) in sitafloxacin group and 97.1% (68/70) in levofloxacin group for the patients with acute uncomplicated UTI corresponding to the bacterial eradication rate of 97.1% (34/35) and 97.6% (41/42) (all p > .05), respectively. For the patients with complicated UTI, the clinical cure rate was 81.8% (27/33) in sitafloxacin group and 76.9% (20/26) in levofloxacin group corresponding to the bacterial eradication rate of 93.3% (14/15) and 63.6% (7/11) (all p > .05), respectively. Sitafloxacin and levofloxacin showed similar incidence of drug-related adverse events. CONCLUSIONS: Oral sitafloxacin is as effective and safe as levofloxacin in treating acute uncomplicated and complicated UTI. KEY MESSAGE: Oral sitafloxacin showed similar clinical cure rate and bacterial eradication rate as levofloxacin for treatment of complicated and uncomplicated urinary tract infections (UTIs) in a randomized, active-controlled, multicentre clinical trial. Oral sitafloxacin is safe and well-tolerated in treating acute uncomplicated and complicated UTIs in Chinese adults. Sitafloxacin is a promising alternative treatment option for UTIs in adults.

[16-gauge vs 18-gauge puncture needle for transrectal ultrasound-guided prostate biopsy in the diagnosis of prostate cancer: Effect and complications].

OBJECTIVE: To compare the efficiency and complications of transrectal ultrasound (TRUS)-guided prostate biopsy with a 16-gauge (16G) or an 18G puncture needle in the diagnosis of PCa. METHODS: This prospective randomized controlled study included 142 male patients undergoing TRUS-guided prostate biopsy in our hospital, 71 with the 16G and the other 71 with the 18G puncture needle. We compared the post-puncture incidence rates of hematuria, bleeding and infection between the two groups of patients and classified the complications according to the Clavien-Dindo scores. RESULTS: The detection rate of PCa was significantly lower in the 18G than in the 16G group (12.68% vs 36.62%, χ2 = 10.958, P = 0.001), even with f/tPSA ≤ 0.15 (8.51% vs 44.44%, χ2 = 12.617, P = 0.001), but showed no statistically significant difference between the two groups with f/tPSA > 0.15 (P<0.05). No post-puncture infection was observed in any of the patients. There were no statistically significant differences between the 18G and 16G groups in the incidence rates of rectal bleeding (21.13% vs 15.49%, χ2 = 0.753, P = 0.385) and urethral bleeding (18.31% vs 16.90%, χ2 = 0.049, P = 0.826), nor in Clavien-Dindo grades (26 vs 20 cases of grade I; no grade II in either group; 2 vs 3 cases of grade III ; Z = －0.698, P = 0.458). CONCLUSIONS: The 16G puncture needle can achieve a higher detection rate of PCa than the 18G needle in TRUS-guided prostate biopsy without increasing the incidence of complications.

Elevated MTA1 induced the migration and invasion of renal cell carcinoma through the NF-κB pathway.

BACKGROUND: The metastasis-associated gene 1 (MTA1) has been extensively reported as a crucial oncogene, and its abnormal expression has been associated with the progression of numerous cancers. However, the role of MTA1 in renal cell carcinoma (RCC) progression and metastasis remains unclear. Herein, we investigated the expression of MTA1 and its role in RCC. METHODS: 109 matched clear cell RCCs (ccRCCs) and corresponding normal tissue samples were analyzed via immunohistochemistry to test the expression of MTA1. Human A498 cell lines were transfected with pcDNA3.1-Flag (control) or Flag-MTA1 to overexpress MTA1 or with specific interfering RNA (si-MTA1) or specific interfering negative control to knockdown MTA1 expression. Transfected cells were used in wound healing and transwell invasion assay. Quantitative real time polymerase chain reaction was used to assess the effect of MTA1 on MMP2/MMP9 and E-cadherin gene expression. Western blot was used to qualify the phosphorylation of p65. RESULTS: Herein, we found a significantly increased expression of MTA1 in 109 ccRCCs, compared to the corresponding normal tissue. In addition, the overexpression of MTA1 in A498 cells facilitated cell migration and invasion, while the down-regulation of MTA1 expression using specific interfering RNA sequences could decrease cell migration and invasion. Furthermore, we showed that MTA1 is up-regulated in ccRCCs, which contributes to the migration and invasion of human kidney cancer cells by mediating the expression of MMP2 and MMP9 through the NF-κB signaling pathway. Similarly, we found that MTA1 could regulate E-cadherin expression in RCCs. CONCLUSIONS: MTA1 is overexpressed in RCC and is involved in the progression of RCC through NF-κB.

The long non-coding RNA LINC00473 contributes to cell proliferation via JAK-STAT3 signaling pathway by regulating miR-195-5p/SEPT2 axis in prostate cancer.

Prostate cancer is a kind of male malignant tumor, which has brought tremendous health threat to men. Prostate cancer is difficult to be cured because of high incidence and metastasis rate. Thereby, it is of great urgency to elucidate the underlying molecular mechanism of prostate cancer for the treatment of this cancer. LINC00473 dysregulation has been observed in many cancers. However, the role of LINC00473 was unknown in prostate cancer. In the present study, we discovered that prostate cancer cells presented high expression of LINC00473, and LINC00473 inhibition limited cell proliferation and the expression of proteins in JAK-STAT3 signaling pathway. Additionally, LINC00473 acted as an up-stream factor for miR-195-5p to negatively modulate miR-195-5p expression. Moreover, SEPT2 interacted with miR-195-5p in prostate cancer and SEPT2 expression was positively modulated by LINC00473 and negatively regulated by miR-195-5p. Last, the inhibitory effect of LINC00473 knockdown on cell proliferation and expression of proteins of JAK-STAT3 signaling pathway was restored by SEPT2 overexpression. All in all, LINC00473 contributed to cell proliferation via JAK-STAT3 signaling pathway by regulating miR-195-5p/SEPT2 axis in prostate cancer, which provided a novel therapeutic tactic for prostate cancer patients.

CTCF-induced upregulation of HOXA11-AS facilitates cell proliferation and migration by targeting miR-518b/ACTN4 axis in prostate cancer.

BACKGROUND: Testified as crucial participators in different types of human malignancies, long noncoding RNAs (lncRNAs) have been revealed to exert a significant effect on the complicated courses of tumor progression. Although existing literatures have revealed the oncogenic role of lncRNA homeobox A11 antisense RNA (HOXA11-AS) in multiple cancers, the underlying role of HOXA11-AS in prostate cancer (PCa) and its potential molecular mechanism remains poorly understood. AIM: To decipher the molecular performance of HOXA11-AS in PCa. METHODS: The expression of HOXA11-AS, miR-518b and actinin alpha 4 (ACTN4) was detected by a real-time quantitative polymerase chain reaction. Colony formation, EdU, flow cytometry, wound healing, and transwell assays were utilized to explore the biological role of HOXA11-AS in PCa. The interaction between RNAs (CCCTC-binding factor [CTCF], HOXA11-AS, miR-518b, and ACTN4) was tested via chromatin immunoprecipitation, luciferase reporter and RNA immunoprecipitation assays. RESULTS: HOXA11-AS in PCa cells was expressed at high levels. Silenced HOXA11-AS in PCa cells could lead to a significant elevation in the abilities of cell proliferation and migration whereas a remarkable declination in cell apoptosis capability. Subsequent molecular mechanism assays confirmed that HOXA11-AS bound with miR-518b and negatively regulates miR-518b expression. Besides, HOXA11-AS could regulate the expression of ACTN4 by sponging miR-518b. Moreover, rescued-function assays revealed that miR-518b inhibition or ACTN4 upregulation reversed the repressive effect of HOXA11-AS knockdown on PCa progression. Furthermore, CTCF was validated to activate HOXA11-AS transcription in PCa cells. CONCLUSIONS: CTCF-induced upregulation of HOXA11-AS facilitates PCa progression via miR-518b/ACTN4 axis, providing a new target for PCa treatment.

Long-term exposure to 4G smartphone radiofrequency electromagnetic radiation diminished male reproductive potential by directly disrupting Spock3-MMP2-BTB axis in the testes of adult rats.

The correlation between long-term exposure to SRF-EMR and the decline in male fertility is gradually receiving increasing attention from the medical society. While male reproductive organs are often exposed to SRF-EMR, little is currently known about the direct effects of long-term SRF-EMR exposure on the testes and its involvement in the suppression of male reproductive potential. The present study was designed to investigate this issue by using 4G SRF-EMR in rats. A unique exposure model using a 4G smartphone achieved localized exposure to the scrotum of the rats for 6 h each day (the smartphone was kept on active talk mode and received an external call for 1 min over 10 min intervals). Results showed that SRF-EMR exposure for 150 days decreased sperm quality and pup weight, accompanied by testicular injury. However, these adverse effects were not evident in rats exposed to SRF-EMR for 50 days or 100 days. Sequencing analysis and western blotting suggested Spock3 overexpression in the testes of rats exposed to SRF-EMR for 150 days. Inhibition of Spock3 overexpression improved sperm quality decline and alleviated testicular injury and BTB disorder in the exposed rats. Additionally, SRF-EMR exposure suppressed MMP2 activity, while increasing the activity of the MMP14-Spock3 complexes and decreasing MMP14-MMP2 complexes; these results were reversed by Spock3 inhibition. Thus, long-term exposure to 4G SRF-EMR diminished male fertility by directly disrupting the Spock3-MMP2-BTB axis in the testes of adult rats. To our knowledge, this is the first study to show direct toxicity of SRF-EMR on the testes emerging after long-term exposure.

Safety parameters of in vivo low-energy shock waves and their impact on adult rat testes.

Low-energy shock waves (LESWs) have been widely used in the intervention of a subset of diseased tissues and organs with good results. However, it is unclear whether they can be used directly to intervene in the testes. Therefore, the aim of this study was to determine a relatively safe energy density and impulse number for rat testes. A total of 176 male rats were randomly and equally assigned to different intervention groups. Among them, 144 rats were assigned to 18 shock subgroups with different energy densities (0.02, 0.04 and 0.06 mJ/mm2), different impulse numbers (500, 1000 and 1500 impulses) and different shock periods (2 and 8 weeks). The remaining 32 rats were divided into the sham intervention (S) groups and the blank control (N) groups with observation periods of 2 weeks and 8 weeks. One day after the last LESWs intervention, all the rats were weighed, and the concentrations of reproductive endocrine hormones were measured, the semen quality and testicular tissue oxidative stress levels were analyzed, and histomorphology and ultrastructures were observed. We found that there were no significant differences in the whole-body physiological state, testicular tissue morphology, oxidative stress state and sperm quality between the L1 shock group and the corresponding S group and N group (all p˃0.05, respectively). However, the other parameters of the shock groups caused different degrees of damage to the structure and function of rat testes, and the whole-body physiological state was also adversely affected. This study demonstrated that LESWs with an energy density of 0.02 mJ/mm2 and 500 impulses had no adverse effects on the rat testes.

Decreased expression of apoptosis-inducing factor in renal cell carcinoma is associated with poor prognosis and reduced postoperative survival.

Apoptosis-inducing factor (AIF) serves a crucial role in cell death and is involved in several types of cancer, including kidney cancer. The present study aimed to explore the association between AIF expression and patient survival based on tumor grades. AIF expression in 96 patients with renal cell carcinoma (RCC) was investigated using immunohistochemistry. Negative AIF expression was determined in 80 patients (83.3%). mRNA expression of AIF was analyzed in RCC and adjacent tissue samples from 15 patients. AIF mRNA expression in RCC tissues were significantly lower compared with that in adjacent tissues. Analysis of histopathological grades revealed that AIF expression was negatively associated with RCC grade, with AIF expression in Grade II tumors being lower than Grade I types, but higher than Grade III. Finally, 68 patients were followed up for 6-118 months, and it was revealed that the overall postoperative survival rate of patients with negative AIF expression was significantly lower compared with those those with positive AIF expression. These results suggest that decreased AIF expression could be associated with worsening RCC grade. Therefore, reduced AIF expression may potentially help diagnose RCC and distinguish tumor grades.

The protective effect of low-energy shock wave on testicular ischemia-reperfusion injury is mediated by the PI3K/AKT/NRF2 pathway.

AIMS: Testicular ischemia-reperfusion (IR) injury is the primary pathophysiological consequence of testicular torsion. Low-energy shock wave (LESW) is an effective treatment for certain diseases. The present study investigated whether LESW could improve on testicular IR injury in rats and examined the involved mechanism. MAIN METHODS: Testicular reperfusion was induced in rats after 1-h ischemia. The first LESW treatment was performed 30 min prior to testicular reperfusion, and then every other day for another 3 applications. LY294002 was applied to investigate the involved mechanism. Testicular morphological changes and malonaldehyde (MDA) level were respectively assessed by hematoxylin-eosin staining. Western blot and thiobarbituric acid method. Western blot, real-time quantitative PCR and immunohistochemistry were performed to assess the apoptosis, the activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) pathway the nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor A (VEGF-A) level in the testis of rats. KEY FINDINGS: LESW improved testicular IR injury in rats. Moreover, LESW upregulated the phosphorylation levels of AKT and glycogen synthase kinase 3ß (GSK-3ß). Also, it upregulated the levels of nuclear NRF2, heme oxygenase 1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO-1) in these rats. Nevertheless, LY294002 blocked these protective effects. LESW also upregulated VEGF-A level in rats with testicular IR injury. SIGNIFICANCE: This study demonstrated that LESW could ameliorate testicular IR injury in rats, which might be attributed to the activation of PI3K/AKT/NRF2 pathway. These findings suggested the potential of LESW in the treatment of testicular torsion.