ObjectiveThis paper aims to study the potential active compound components and action mechanism of Shenqi Dihuangtang in the treatment of diabetic kidney disease （DKD） through network pharmacology and in vivo experimental verification. MethodsUltra-high-performance liquid chromatography-Q-exactive orbitrap mass spectrometry （UPLC-Q-Exactive Orbitrap MS） technology was used to clarify the main active chemical components of Shenqi Dihuangtang， and it was combined with network pharmacology methods such as gene ontology （GO） functional annotations and Kyoto encyclopedia of genes and genome （KEGG） to predict the potential action mechanism of Shenqi Dihuangtang in treating DKD. Subsequently， the DKD model of db/db male mice was established， and the mice were randomly divided into model group， low-dose Shenqi Dihuangtang group （6.10 g·kg^-1）， medium-dose Shenqi Dihuangtang group （12.19 g·kg^-1）， high-dose Shenqi Dihuangtang group （24.38 g·kg^-1）， and daplizin group （1.25 mg·kg^-1）. During the same period， C57BL/6J male mice were selected into normal group and received drug intervention for 8 weeks， respectively. During this period， the body weight and fasting blood glucose （FBG） of the mice were dynamically monitored， and oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were performed at the end of dosing. The levels of serum creatinine （SCr）， blood urea nitrogen （BUN）， uric acid （UA）， albumin （ALB）， and total protein （TP） were measured by an automatic biochemical analyzer， and 24-hour urine protein was measured by a urine protein quantitative kit. Hematoxylin-eosin （HE）， periodic-acid Schiff （PAS）， and Masson staining were employed to observe the renal histopathology. The expression of nephrotic protein Nephrin was observed by immunohistochemistry. Western blot was used to detect the expression of epithelial-mesenchymal transition （EMT）-related proteins such as TGF-β₁， Smad2/3， α-smooth muscle actin （α-SMA）， neural-cadherin （N-Cadherin）， and snail protein. ResultsUPLC-Q-Exactive Orbitrap MS identified 384 active compounds in the aqueous extract of Shenqi Dihuangtang. According to oral bioavailability≥30% and the five drug-like principles， 44 key active ingredients were screened out， and 169 intersection targets highly correlated with DKD were matched. Among them， there was a significant interaction relationship between tumor necrosis factor（TNF）， interleukin（IL）-6， protein kinase B（Akt）1， Caspase-3， Jun proto-oncogene （JUN）， hypoxia inducible factor-1α（HIF-1α）， B cell lymphoma-2（Bcl-2）， matrix metallopeptidase-9（MMP-9）， IL-1β， and TGF-β₁. GO functional annotations were significantly enriched in cellular components such as membrane rafts， membrane microdomains， and collagen-containing extracellular matrix， molecular functions such as DNA-binding transcription factor binding， R-Smad binding， and Smad protein binding， as well as biological processes such as reactions to lipopolysaccharides（LPS）， reactions to bacteria-derived molecules， and wound healing. The KEGG pathway was significantly enriched in lipids and atherosclerosis， TGF-β signaling pathway， phosphatidylinositol 3 kinase （PI3K）/Akt signaling pathway， etc. In vivo experimental results showed that the high-dose Shenqi Dihuangtang group could significantly reduce FBG levels in db/db mice （P<0.01）， improve OGTT （P<0.01） and ITT （P<0.01） levels， reduce SCr （P<0.01）， BUN （P<0.01）， UA （P<0.01） and 24-hour BUN （P<0.01）， and increase ALB （P<0.01） and TP （P<0.01） levels. Pathological staining confirmed that the high-dose Shenqi Dihuangtang group could significantly reduce the glomerular mesangial matrix area and collagen deposition （P<0.01） and upregulate the positive expression rate of Nephrin （P<0.01）. Western blot results showed that the high-dose Shenqi Dihuangtang group significantly downregulated the expression of TGF-β₁ （P<0.01） and Smad2/3 （P<0.01） signal molecules and inhibited the protein levels of α-SMA （P<0.01）， N-Cadherin （P<0.01）， and Snail （P<0.01）. ConclusionShenqi Dihuangtang can inhibit the TGF-β₁/Smad signaling axis and block the renal EMT process， thereby improving DKD renal fibrosis damage. Further analysis of its key active components and clinical transformation pathways is needed in the future.

Objective To compare the early clinical efficacy of arthroscopic bridge suture combined with strengthening thread fixation for posterior cruciate ligament(PCL)tibial insertion avulsion frac-tures and traditional open double-row anchor suture fixation,and to explore a safe,effective,and min-imally invasive reduction and fixation method for PCL tibial insertion fractures in knee joint under ar-throscopy.Methods This study was a retrospective study.Sixty patients with PCL tibial insertion frac-tures(Meyers&McKeever types II-IV)and admitted to the Department of Orthopedics of Kunming Hospital of Traditional Chinese Medicine between June 2021 and June 2023 were included,and ran-domly divided into a traditional group(age:39.56±3.24 years,male/female:16/14)and a minimally in-vasive group(age:40.32±4.38,male/female:18/12),each of 30,according to their admission or-der.All patients were of Grade III positive in the drawer test before surgery.The traditional group un-derwent posterior medial inlay incision,reduction under direct vision and double-row anchor suture fix-ation,while the minimally invasive group underwent reduction under total arthroscopy,suture-bridge suture at the tendon-bone junction of PCL,and one anchor suture placed at the femoral insertion of PCL,with the anchor suture line serving as the reinforcing suture.Then,the anchor and suture-bridge suture lines were respectively pulled into the two tunnels behind the tibia from the front and then pulled out from the front of the tibia,and the reinforcing suture was knotted in front of the tib-ia.The bridge suture line was fixed in front of the tibia with an external row anchor.Six months after surgery,both groups were evaluated the efficacy using the posterior drawer test and Lysholm score.Re-sults At the follow-up six months after the surgery,both groups had good fracture healing,with one wound infection and delayed healing in the traditional group and total one-stage heal in the other group.According to the physical examination 6 months after surgery,all were negative in the posterior drawer test,except one of degree II positive in the traditional group.Moreover,the average Lysholm score of the traditional group increased from 65.23±3.48 before surgery to 87.64±4.58 points after surgery(P<0.05),while that of the minimally invasive group increased from 64.35±2.52 to 86.82±2.58(P<0.05),showing no significant differences between the two groups(P>0.05).Conclusion Both techniques in this study can achieve excellent surgical outcomes in the treatment of posterior cruciate ligament avulsion fractures at the tibial insertion site.

Objective To compare the early clinical efficacy of arthroscopic bridge suture combined with strengthening thread fixation for posterior cruciate ligament(PCL)tibial insertion avulsion frac-tures and traditional open double-row anchor suture fixation,and to explore a safe,effective,and min-imally invasive reduction and fixation method for PCL tibial insertion fractures in knee joint under ar-throscopy.Methods This study was a retrospective study.Sixty patients with PCL tibial insertion frac-tures(Meyers&McKeever types II-IV)and admitted to the Department of Orthopedics of Kunming Hospital of Traditional Chinese Medicine between June 2021 and June 2023 were included,and ran-domly divided into a traditional group(age:39.56±3.24 years,male/female:16/14)and a minimally in-vasive group(age:40.32±4.38,male/female:18/12),each of 30,according to their admission or-der.All patients were of Grade III positive in the drawer test before surgery.The traditional group un-derwent posterior medial inlay incision,reduction under direct vision and double-row anchor suture fix-ation,while the minimally invasive group underwent reduction under total arthroscopy,suture-bridge suture at the tendon-bone junction of PCL,and one anchor suture placed at the femoral insertion of PCL,with the anchor suture line serving as the reinforcing suture.Then,the anchor and suture-bridge suture lines were respectively pulled into the two tunnels behind the tibia from the front and then pulled out from the front of the tibia,and the reinforcing suture was knotted in front of the tib-ia.The bridge suture line was fixed in front of the tibia with an external row anchor.Six months after surgery,both groups were evaluated the efficacy using the posterior drawer test and Lysholm score.Re-sults At the follow-up six months after the surgery,both groups had good fracture healing,with one wound infection and delayed healing in the traditional group and total one-stage heal in the other group.According to the physical examination 6 months after surgery,all were negative in the posterior drawer test,except one of degree II positive in the traditional group.Moreover,the average Lysholm score of the traditional group increased from 65.23±3.48 before surgery to 87.64±4.58 points after surgery(P<0.05),while that of the minimally invasive group increased from 64.35±2.52 to 86.82±2.58(P<0.05),showing no significant differences between the two groups(P>0.05).Conclusion Both techniques in this study can achieve excellent surgical outcomes in the treatment of posterior cruciate ligament avulsion fractures at the tibial insertion site.

Objective:To explore the clinical and genetic features of two Chinese pedigrees affected with Autosomal dominant intellectual developmental disorder 49 (MRD49).Methods:Two MRD49 pedigrees which were admitted to the Children′s Hospital Affiliated to Shandong University respectively on January 28, 2021 and November 10, 2022 were selected as the study subjects. Clinical data of the two pedigrees were collected and analyzed. Genomic DNA was extracted from peripheral blood samples of the probands and their family members. The probands were subjected to mutational analysis by high-throughput sequencing. Candidate variants were validated using real-time fluorescence quantitative PCR (q-PCR) or Sanger sequencing and bioinformatic analysis. This study was approved by the Medical Ethics Committee of the Children′s Hospital Affiliated to Shandong University (No. SDFE-IRB/T-2022002).Results:Proband 1 had presented with language delay, motor retardation and intellectual disability, and his maternal grandmother, mother, aunt and cousin all had various degrees of intellectual disability. Sequencing results showed that proband 1 had deletion of exons 3 ~ 7 of the TRIP12 gene. q-PCR verification showed that his mother, aunt, maternal grandmother and cousin had all harbored the same deletion. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PVS1+ PM2_Supporting+ PP1). Proband 2, who had mainly presented with language delay, motor retardation and intellectual disability, and was found to harbor a heterozygous c.3010C>T (p.Arg1004*) variant of the TRIP12 gene, which was verified to be de novo in origin. Based on the guidelines from the ACMG, the variant was classified as pathogenic (PVS1+ PS2+ PM2_Supporting). Conclusion:This study had diagnosed two MRD49 families through high-throughput sequencing. Above findings have enriched the phenotypic and mutational spectrum of MRD49 in China, which has also facilitated genetic counseling for the two pedigrees.

Background Environmental pollutants can affect N⁶-methyladenosine (m⁶A) level in the body, but the change of m⁶A level in kidney after being exposed to cadmium (Cd) and the molecular mechanism of renal injury need to be further studied. Objective To analyze the associations of m⁶A modification and methyltransferases/demethylases with microRNA-21 (miR-21) and transforming growth factor- β1 (TGF - β1) in kidney of rats exposed to Cd. Methods Twenty-four SPF male SD rats were divided into 4 groups, with 6 rats in each group, and were exposed to Cd by subcutaneous injection of 2.0, 1.0, and 0.5 mg·kg⁻¹ cadmium chloride (CdCl₂) and equal volume of normal saline for 2 weeks, 7 d a week, respectively. The levels of N-acetyl-β-D-glucosidase (UNAG) and albumin (UALB) in urine, and the levels of m⁶A methylation and TGF-β1 in kidney were detected by enzyme-linked immunosorbent assay (ELISA). The level of blood urea nitrogen (BUN) was measured by urease method. The levels of renal oxidative stress indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were detected by total bile acid method, water-soluble tetrazolium asssay, and colorimetric method respectively. The relative levels of TGF-β1, methyltransferases, and demethylases in kidney were measured by reverse transcription-polymerase chain reaction. The expression of miR-21 in kidney was detected by fluorescent quantitative polymerase chain reaction. Results After 2 weeks of exposure to Cd, the body weights of rats in the 2.0 and 1.0 mg·kg⁻¹ cadmium chloride groups decreased, and the ratio of kidney/body weight and the levels of BUN, UNAG, and TGF-β1 mRNA and protein increased in the 2.0 mg·kg⁻¹ cadmium chloride group (P<0.05). The expression levels of m⁶A modification, methyltransferases METTL3, METTL14, Wilms’ tumor 1-associated protein (WTAP), and miR-21 were increased both in the 2.0 and 1.0 mg·kg⁻¹ cadmium chloride groups, with significant differences compared with the control group (P<0.05). The results of correlation analysis showed that the m⁶A modification level was negatively correlated with SOD (r=−0.4489, P<0.05) and GSH-Px (r=−0.4874, P<0.05), METTL3 was negatively correlated with MDA (r=−0.5158, P<0.05), while there was a positive correlation between FTO and GSH-Px (r=0.4802, P<0.05). In addition, miR-21 was positively correlated with METTL3 (r=0.7491), METTL14 (r=0.6157), and WTAP (r=0.6660) (P<0.05), TGF-β1 was positively correlated with METTL3 (r=0.5025, P<0.05) but negatively correlated with FTO (r=−0.5634, P<0.05) . Conclusion Cd can induce m⁶A methylation and up-regulation of METTL3, METTL14, WTAP, and miR-21 expression levels in rat kidney tissues, indicating that m⁶A and miR-21 may be associated with Cd-induced renal fibrosis.

OBJECTIVETo explore the influences of di-(2-ethylhexyl)phthalate (DEHP) and its principle metabolite mono(2-ethylhexyl)phthalate (MEHP) on spermatogenic cell apoptosis in young male Wistar rats.

METHODSNinety-eight 2-week-old male Wistar rats were randomly divided into 14 equal groups to receive daily intragastric administration of 0.2 ml/kg normal saline for 3 weeks (normal control), 100 mg/kg cyclophosphamide (CTX) for 1 week (positive control), 100, 200, and 300 mg/kg DEHP or MEHP for 1 week, or 100 mg/kg DEHP or MEHP for 1, 2, and 3 weeks. After the treatments, the pathological changes of the testicular tissues were examined, spermatogenic cell apoptosis was detected, and serum sex hormones levels were measured using TUNEL assay or radioimmunoassays.

RESULTSCTX, DEHP, and MEHP all caused shrinkage, development retardation and quantitative reduction of spermatogenic cells with and mitochondrial swelling vacuolar changes. The damage of spermatogenic cells increased significantly with the increment of DEHP and MEHP doses and exposure time. Both DEHP and MEHP treatments resulted in significantly increased cell apoptosis index (AI) in close correlation with the exposure doses and duration (P<0.01). DEHP and MEHP treatments also significantly increased serum levels of follicle stimulating hormone and luteinizing hormone and decreased testosterone levels in a dose- and time-dependent manner (P<0.05).

CONCLUSIONDEHP and MEHP can induce obvious apoptosis of spermatogenic cells in young male rats with a dose- and time-dependent effect.

Animals ; Apoptosis ; drug effects ; Diethylhexyl Phthalate ; analogs & derivatives ; toxicity ; Dose-Response Relationship, Drug ; Environmental Exposure ; Male ; Rats ; Rats, Wistar ; Spermatozoa ; cytology ; drug effects