This article summarizes professor LIU Guangzhen's clinical experience in treating diabetic kidney disease （DKD） using the"four methods of unblocking collaterals". It is believed that the disease location of DKD centers around renal collaterals， with wind-damp turbidity and stasis obstructing the renal collaterals being the core pathogenesis. The four methods of unblocking collaterals are proposed to treat the disease which include expelling wind， dispelling dampness， draining turbid and dissolving stasis to unblock collaterals， and Tangshen Tongluo Formula （糖肾通络方） is designed as a basic prescription. In clinical practice， when applying the four methods of unblocking collaterals， it is necessary to adjust the treatment according to the dominance of wind， dampness， turbid， or stasis， and to flexibly choose the appropriate medication based on syndrome differentiation.

Objective: To elucidate the potential mechanisms by which Chinese herbal medicine (CHM) regulates gut microbiota (GM) to influence the development of primary open-angle glaucoma (POAG). Methods: Data mining, network pharmacology, and Mendelian randomization (MR) analyses (two-sample design) were conducted in integration to systematically explore the CHM-GM-POAG axis. Literature-based data mining method was applied to identify frequently used herbs and herb pairs for POAG, and the properties and meridian tropism of the herbs were analyzed as well. Target prediction and pathway enrichment analyses were performed to identify shared molecular pathways among CHM components, GM, and POAG. MR analysis was performed to assess the genetically predicted causal associations between specific microbial taxa and POAG risk. Results: Our data mining work indicated that commonly used CHMs were mainly bitter and sweet in flavors and cold in property, with meridian tropism toward the liver, lung, and kidney. The predominant therapeutic effects of the CHMs included soothing the liver and regulating Qi, promoting blood circulation, and reducing fluid retention. Representative herb pairs were Shudihuang (Rehmanniae Radix Praeparata)-Gouqi (Lycii Fructus) with Zexie (Alismatis Rhizoma), Gouqi (Lycii Fructus)-Fuling (Poria) with Shudihuang (Rehmanniae Radix), and Juhua (Chrysanthemi Flos)-Gouqi (Lycii Fructus) with Zexie (Alismatis Rhizoma). Network pharmacology revealed overlapping targets involving antioxidative, anti-inflammatory, and metabolic regulation pathways. MR analysis demonstrated that higher abundances of Ruminiclostridium 6 [odds ratio (OR) = 0.73, 95% confidence interval (CI): 0.58 – 0.92, P = 0.007], Ruminococcaceae UCG-002 (OR = 0.77, 95% CI: 0.63 – 0.96, P = 0.018), Ruminococcus torques group (OR = 0.71, 95% CI: 0.57 – 0.90, P = 0.004), and Victivallis (OR = 0.82, 95% CI: 0.70 – 0.96, P = 0.016) were causally associated with reduced POAG risk, whereas Actinomyces (OR = 1.34, 95% CI: 1.06 – 1.68, P = 0.013) and Blautia (OR = 1.39, 95% CI: 1.01 – 1.90, P = 0.042) showed positive associations. Conclusion This study revealed potential causal links between GM and POAG and provided integrative evidence that CHM may modulate the microbiota to exert neuroprotective effects. These findings offer new integrative insights into the gut-eye axis and a theoretical basis for developing microbiota-targeted CHM strategies in glaucoma management.

Objective To investigate the effect and underlying mechanism of Qingguang’an Granules (青光安颗粒剂, QGAG) on mitochondrial autophagy (mitophagy) of retinal ganglion cells (RGCs) in rats with chronic ocular hypertension (COH). Methods Sixty Sprague Dawley (SD) rats, half males and half females, were randomly assigned to three groups: the control, model, and QGAG (2.5 g/kg) groups, with 20 rats in each group. Rats’ model of COH was established by cauterizing episcleral veins in the model group and QGAG group. Three weeks after successful modeling, rats in the QGAG group were intragastrically administered with QGAG, while rats in the control group and the model group received an equal dose of normal saline. After three months of intragastric administration, intraocular pressure (IOP) of all rats was measured. The mitophagy was monitored by the immunofluorescence method, the mitochondrial membrane potential was measured using the JC-1 method, and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy. Meanwhile, rat RGCs were labeled using the fluorescent gold method, and RGCs density in each group was calculated. Moreover, RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. Finally, the expression levels of Parkin, optineurin, microtubule-associated protein 1 light chain 3-II/microtubule-associated protein 1 light chain 3-I (LC3-II/LC3-I), recombinant lysosomal associated membrane protein 1 (LAMP1), and B-cell lymphoma-2 (Bcl-2) in RGCs were determined by Western blot assay. The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction (qRT-PCR). Results The QGAG reduced IOP in COH rats, and inhibited mitophagy and apoptosis of RGCs (P < 0.05). Besides, the QGAG significantly increased the expression levels of Parkin and Bcl-2 (P < 0.05), and inhibited the expression levels of optineurin, LAMP1, and LC3-II/LC3-I (P < 0.05) in RGCs of COH rats. Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage caused by glaucoma, which may be mediated through the mitophagy ubiquitination via the Parkin/PINK1-related pathway.

This study was aimed to explore a simple and applicable method of separating mature sperms from human testicular tissue for intracytoplasmic sperm injection and embryo transfer. The suspension of human testicular tissue was cultured in 10% human serum albumin and human tubule fluid with different concentrations (0 u/ml; 50 u/ml; 100 u/ml; 150 u/ml; 200 u/ml) of hyaluronidase for 24 h, and then the Percoll gradient centrifugation was processed to separate the sperms; meanwhile the sperms were counted and graded according to their motility. The difference in quality and quantity among the groups and the difference between the groups and the zero-hour culturing group were detected. It was shown that the four hyaluronidase-treated groups contained large quantity and high quality of sperms as compared with the two contrast groups (P<0.01). The groups in the solution of 50 u/ml, 100 u/ml and 150 u/ml concentrations of hyaluronidase had almost the same amount of sperms that displayed higher motility as compared against the sperms in the group treated with 200 u/ml concentration of hyaluronidase (P<0.01). There was no difference between the two contrast groups (P>0.05), or among the groups treated with 50 u/ml, 100 u/ml, and 150 u/ml of hyaluronidase concentration (P>0.05). This method of adopting hyaluronidase with Percoll gradient centrifugation in the process for separating mature sperms from human testicular tissue is applicable. It can increase the quantity and quality of sperms separated from testicular tissue suspensions when adequate concentrations of hyaluronidase is used.
Adult ; Cell Separation ; methods ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Hyaluronoglucosaminidase ; pharmacology ; Male ; Spermatozoa ; cytology ; Testis ; cytology

Objective:To investigate the effects of lentinan combined with HCV NS3 DNA vaccine on the antigen specific immune response.Methods:BALB/c mice were ip with different dose of lentinan plus the same dose of NS3 plasmid(100 ?g/per mouse) in a prime and boost immunization strategy.10 days after the second injection,spleens were taken.The cell suspension was prepared and cultured with NS3 peptide as the stimulant.In addition,NS3 cocultured P815 cells were used as target cells.NS3 specific T cell proliferation and cytotoxicity T lymphocyte reaction(CTL)were analyzed by MTT methods,and the cytokines IL-4 and IFN-? in serum were detected by ELISA technique.Results:It was showed that the high and middle dose of lentinan plus NS3 plasmid specifically stimulated NS3 specific CTL reaction at the ratio of effector to target as 50∶1,compared with the single use of NS3 plasmid,P