The BV2 cells used in the present study are immortalized microglia and were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). BV2 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China), 100 U/ml penicillin and 100 µg/ml streptomycin (Biosharp, Hefei, China). The BV2 cells were incubated in a cell incubator with a humidified atmosphere of 95% air and 5% CO₂ at 37°C.

miR-195 mimics (5′-UAGCAGCACAGAAAUAUUGGC-3′), miRNA mimics negative control (miR-NC1; 5′-UCACAACCUCCUAGAAAGAGUAGA-3′), miR-195 inhibitor (5′-GCCAAUAUUUCUGUCUGCUA-3′) and miRNA inhibitor negative control (miR-NC2; 5′-UCACAACCUCCUAGAAAGAGUAGA-3′) were purchased from Guangzhou RiboBio Co., Ltd., (Guangzhou, China). ROCK1 small interfering (si)RNA (siROCK1; cat. no. sc-36432) and control siRNA (cat no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cell transfection was performed when cell confluence reached 75%. Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for cell transfection according to the manufacturer's protocols. After 6 h, the transfection mixture was replaced with fresh culture medium and the cells used for subsequent experimentation. The concentration of miR-195 mimics, miR-195 inhibitor and ROCK1 siRNA was 100 nM.

Total RNA was extracted from BV2 cells using TRIzol^® reagent according to the protocol provided by the manufacturer (Thermo Fisher Scientific, Inc.). RT-qPCR was performed according to a previously described protocol (15). U6 and GAPDH were used as the invariant controls and the mRNA expression of target genes was expressed as fold changes following normalization to GAPDH or U6. The primers used are summarized in Table I.

Microglia cells were treated with 1 µg/ml LPS for 24 h and transfection with 100 nM miR-195 mimics for another 6 h. The levels of inducible nitric oxide synthase (iNOS; cat. no., A014-1-1), interleukin (IL)-6 (cat. no. H007), tumor necrosis factor-α (TNF-α; cat. no. H052), IL-4 (cat. no. H005) and IL-10 (cat. no. H009) in whole cell lysate of microglia were determined using corresponding ELISA kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the protocols provided by the manufacturer.

The cell proliferation rate was determined using a non-radioactive proliferation assay kit (X1327K; Exalpha Biologicals, Inc., Shirley, MA, USA) according the manufacturer's protocol. The ELISA is able to detect the incorporation of 5-bromo-2-deoxyuridine (BrdU) into newly-synthesized DNA in proliferated cells. BrdU solution was added into the culture system following treatment of the cells with 1 µg/ml LPS for 24 h and transfection with 100 nM miR-195 inhibitor and ROCK1 siRNA for 6 h. Subsequently, cells were cultured for an additional 16 h. The cell medium was removed and the BrdU immunoreactivity was detected using prediluted anti-BrdU Detector Antibody and 1X Peroxidase Goat anti-Mouse IgG included in the kit. The absorbance was detected at 450 and 540 nm. The results were expressed as a fold of the vehicle control.

Cell viability was detected using a Cell Counting Kit-8 (CCK-8) cell viability assay (Nanjing Jiancheng Bioengineering Institute). Cells were cultured in a 96-well plate (1×10⁴/well) and then treated with 1 µg/ml LPS for 24 h and transfection with 100 nM miR-195 inhibitor and ROCK1 siRNA for 6 h. Subsequently, 10 µl CCK-8 solution was added to each well, followed by incubation at 37°C for 1 h. The SpectraMax™ microplate spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA) was used to detect the absorbance at 450 nm.

Cell apoptosis was measured by flow cytometry (FCM) using an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) kit (Vazyme Biotech, Co., Ltd., Nanjing, China). Following treatment with 1 µg/ml LPS for 24 h and transfection with 100 nM miR-195 inhibitor and ROCK1 siRNA for 6 h, BV2 cells were detached from the culture plates with 0.25% trypsin (Beyotime Institute of Biotechnology, Haimen, China) and washed with PBS 3 times. Following removal of the PBS, the cells were suspended in 1 ml binding buffer (Vazyme Biotech, Co., Ltd.) supplemented with 10 µl FITC and PI each. Cells were incubated in the dark for 15 min and then measured by FCM (BD Biosciences, San Jose, CA, USA). Data were analyzed using BD CellQuest Pro software (version 5.1; BD Biosciences, Franklin Lakes, NJ, USA) and expressed as a fold of the vehicle control.

All of the experimental data are expressed as the mean ± standard deviation. The results were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). The significance of differences between groups was determined by one-way analysis of variance followed by Dunnett's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To preliminarily assess the association between miR-195 expression and microglial activation, the expression of miR-195 in ramified quiescent and activated microglia was initially detected. LPS was used to establish an in vitro model of microglia activation. The results indicated that, compared with the control, LPS inhibited miR-195 expression in BV2 cells in a concentration-dependent manner. Notably, no significant difference in miR-195 expression between the 1 and 2 µg/ml LPS-stimulated groups was observed (Fig. 1A). In addition, LPS at the concentration of 1 µg/ml decreased the expression of miR-195 in BV2 cells in a time-dependent manner (Fig. 1B). Taken together, miR-195 expression was decreased in LPS-stimulated BV2 cells in a concentration- and time-dependent manner.

Microglia activation is a complex process accompanied by extensive release of inflammatory factors. Given that the expression of miR-195 was markedly decreased in LPS-induced activated BV2 cells, the function of miR-195 in microglia activation was then investigated. It was confirmed that, compared with the control, transfection with miR-195 mimics markedly increased the level of miR-195 (Fig. 2A), whereas miR-195 inhibitors decreased the level of miR-195 expression (Fig. 2B), suggesting that the gain- or loss-of-function of miR-195 was successfully achieved in the in vitro system. Furthermore, it was revealed that miR-195 mimics inhibited the expression of pro-inflammatory cytokines, including iNOS, IL-6 and TNF-α, but promoted the expression of anti-inflammatory cytokines in LPS-treated BV2 cells, including IL-4 and IL-10 (Fig. 2C-G). However, miR-195 inhibitors enhanced the stimulatory effects of LPS on the expression of these neuroinflammation-associated factors, characterized by an increase in the expression of iNOS, IL-6 and TNF-α, and a decrease in the expression of IL-4 and IL-10 (Fig. 2H-L). Consistent with the qPCR results, the ELISA data indicated similar variations of iNOS, IL-6, TNF-α, IL-4 and IL-10 in microglia following stimulation with LPS and miR-195 mimics or inhibitor (Fig. 2M and N). Taken together, these results demonstrated that miR-195 may have a crucial role in suppressing LPS-induced activation of microglia.

In view of the pivotal role of ROCK1 in modulating inflammation, the present study investigated whether ROCK1 functions downstream of miR-195 in the inflammatory signaling pathways of in PD. The results indicated that, compared with the control, LPS stimulated the mRNA and protein expression of ROCK1 in a concentration- and time-dependent manner (Fig. 3). Of note, miR-195 mimics markedly decreased the mRNA and protein levels of ROCK1, while miR-195 inhibitors enhanced the expression of ROCK1 mRNA and protein (Fig. 4). These results suggest that ROCK1 expression is positively associated with microglia activation but negatively regulated by miR-195 in microglia.

To systematically investigate the intricate regulatory mechanisms, the potential role of ROCK1 in regulating microglia activation was additionally explored. It was confirmed that, compared with the control, siROCK1 markedly decreased the expression of ROCK1, suggesting that ROCK1 in microglia was efficiently knocked down (Fig. 5A). ROCK1 knockdown inhibited LPS-induced activation of BV2 cells, evidenced by decreased cellular iNOS, IL-6 and TNF-α, and increased IL-4 and IL-10 (Fig. 5B-F). Consistent with the qPCR results, the results of the ELISAs demonstrated similar changes in the levels of iNOS, IL-6, TNF-α, IL-4 and IL-10 in microglia following stimulation with LPS subsequent to transfection with siROCK1 (Fig. 5G). Taken together, these results indicated that inhibition of ROCK1 may prevent microglia activation.

The role of the miR-195/ROCK1 pathway in regulating cell proliferation and viability was then investigated. The results indicated that LPS enhanced the proliferative capacity of microglia, and this effect was additionally increased in miR-195 inhibitor-transfected microglia. Notably, ROCK1 knockdown reversed the effects of miR-195 inhibitor (Fig. 6A). Consistently, the results of the cell viability assay exhibited a similar trend (Fig. 6B). Collectively, these data suggest that the miR-195/ROCK1 pathway has a crucial role in modulating the proliferation and viability of BV2 cells challenged with LPS.

To determine whether cell apoptosis was also affected by the miR-195/ROCK1 pathway, the Annexin V-FITC-PI apoptosis detection kit was used and FCM analysis was performed. The results demonstrated that miR-195 inhibitor suppressed the apoptosis of LPS-treated BV2 cells, which, was reversed by knockdown of ROCK1 (Fig. 7). It was therefore indicated that the miR-195/ROCK1 pathway is involved in regulating LPS-induced microglia apoptosis.