cPKCγ Inhibits Caspase-9-Initiated Neuronal Apoptosis in an Ischemia Reperfusion Model In Vitro Through p38 MAPK-p90RSK-Bad Pathway.

Strokes are one of the leading causes of death and disability in the world. Previously we have found that conventional protein kinase Cγ (cPKCγ) plays neuroprotective role in ischemic strokes. Further, we found that cPKCγ knockdown increased the level of cleaved (cl)-Caspase-3. However, the precise mechanisms underlying cPKCγ-mediated neuronal death remain unclear. To this end, a model incorporating 1 h oxygen-glucose deprivation/24 h reoxygenation (1 h OGD/24 h R) was established in cortical neurons. We found that cPKCγ knockdown remarkably increased neuronal death after OGD. We also found that cPKCγ knockdown increased the level of cl-Caspase-3 through the upstream initiators Capsases-9 (not Caspase-8/12) in OGD-treated neurons. Overexpression of cPKCγ could decrease neuronal death and cl-Caspase-3 and -9 levels. Moreover, cPKCγ knockdown further reduced the phosphorylation levels of p38 MAPK, p90RSK, and Bad. In addition, the protein levels of Bcl-2 and Bcl-xl were decreased after cPKCγ knockdown, whereas that of Bax was increased. In conclusion, our results suggest that cPKCγ partly alleviates ischemic injury through activating the p38 MAPK-p90RSK-Bad pathway and inhibiting Caspase-9 initiated apoptosis. This may have potential as a therapeutic target for ischemic stroke.

cPKCγ ameliorates ischemic injury in cultured neurons exposed to oxygen glucose deprivation/reoxygenation by inhibiting ferroptosis.

Ferroptosis is an iron-dependent pathway of regulated cell death. But the exact mechanism of ferroptosis in ischemic stroke remains unclear. We hypothesize that conventional protein kinase cγ (cPKCγ) can attenuate neuronal death by regulating ferroptosis. In this study, primary cultured cortical neurons were used to establish 1 h oxygen-glucose deprivation (OGD) and reoxygenation (R) 0-12 h (i.e., 1 h OGD/R 0-12 h) as in vitro models of cell ischemia. After 1 h OGD/R 0-12 h, cyclooxygenase 2 (COX2) and acyl-CoA synthetase long-chain family member 4 (ACSL4) levels increased, and glutathione peroxidase 4 (GPx4) levels decreased significantly. Concurrently, GPx4 activity decreases, and iron levels increased. The inhibition of ferroptosis by Liproxstatin-1 ameliorated OGD-induced neuronal injury. Liproxstatin-1 administration prominently induced GPx4 expression and suppressed COX2 expression. Additionally, Liproxstatin-1 administration substantially reduced iron accumulation and rescued GPx4 activity, accompanying by prominent changes in lipid peroxidation indicators. cPKCγ knockdown significantly aggravated neuronal death, and increased GPx4 depletion and COX2 and ACSL4 levels, thus dramatically increasing iron accumulation and GPx4 inactivation. Changes in lipid peroxidation indicators were also significantly increased. Ferroptosis is closely associated with OGD-induced ischemic injury, and cPKCγ can attenuate ischemic injury after OGD via ferroptosis suppression.

Downregulation of miR-338-3p alleviates neuronal ischemic injury by decreasing cPKCγ-Mediated autophagy through the Akt/mTOR pathway.

Ischemic stroke is the leading cause of mortality and disability in aging populations. Dysregulation of microRNA is associated with the pathophysiology of ischemic brain injury. Previously, we found that miR-338-3p was prominently downregulated in OGD-treated neurons, which indicates that miR-338-3P potentially plays an important role in ischemic injury. Furthermore, we performed a bioinformatic analysis and found that conventional protein kinase cγ (cPKCγ), an important autophagy regulator, is a potential target of miR-338-3p, and it is upregulated in neurons after ischemic injury. Therefore, we speculated that miR-338-3P may play a role in neuronal autophagy associated with ischemic brain injury by regulating cPKCγ levels. In the present study, oxygen glucose deprivation was used to test this hypothesis. Our results show that miR-338-3p expression is prominently downregulated after OGD. Additionally, miR-338-3p knockdown attenuated ischemic injury and simultaneously reduced the microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio, which contributes to neuronal survival after ischemia. Moreover, the cPKCγ protein level increased, and miR-338-3p recognized the 3'-untranslated region of the cPKCγ messenger RNA (mRNA) and negatively regulated the cPKCγ protein level by promoting the degradation of its mRNA. In addition, Lv-cPKCγ blocked the pri-miR-338-3p-induced decrease of the Akt and mammalian target of rapamycin (mTOR) phosphorylation levels, as well as the accompanying increase of the LC3-II/LC3-I ratio, thereby alleviating ischemic injury. This suggests that miR-338-3p downregulation following ischemic injury alleviates neuronal injury by targeting cPKCγ, thereby activating the Akt/mTOR signaling cascade and decreasing downstream autophagy. These results provide a potential therapeutic target for ischemic stroke.

A tri-functional probe mediated exponential amplification strategy for highly sensitive detection of Dnmt1 and UDG activities at single-cell level.

Multiplex DNA methylation and glycosylation are ubiquitous in the human body to ensure the normal function and stability of the genome. The methyltransferases and glycosylases rely on varied enzymes with different action mechanism, which still remain challenges for multiple detection. Herein, we developed a tri-functional dsDNA probe mediated exponential amplification strategy for sensitive detection of human DNA (cytosine-5) methyltransferase 1 (Dnmt1) and uracil-DNA glycosylase (UDG) activities. The tri-functional dsDNA probe was rationally designed with M-DNA and U-DNA. M-DNA contains the 5'-GCmGCGC-3' site for Dnmt1 recognition. U-DNA possesses one uracil as the substrate of UDG and a primer sequence for initiating the amplification reaction. M-DNA was complementary to partial sequence of U-DNA. In the presence of Dnmt1 and UDG, BssHⅡ and Endo Ⅳ were used to nick the 5'-GCGCGC-3' and AP sites respectively, resulting in the release of single-stranded DNA sequence (primer sequence), respectively. After magnetic separation, the released primer sequence hybridizes with padlock DNA (P-DNA), initiating exponential rolling circle amplification to produce numerous G-quadruplexes for recordable signals. The strategy exhibited the limit of detection as low as 0.009 U mL-1 and 0.003 U mL-1 for Dnmt1 and UDG, respectively. Meanwhile, this strategy was successfully applied to detect Dnmt1 and UDG activities in living cell samples at single-cell level and assay the inhibitors of Dnmt1 and UDG. Therefore, the strategy provided a potential method to detect Dnmt1 and UDG activities in biological samples for early clinic diagnosis and therapeutics.

[Neuroprotective effects of linagliptin on ischemia/reperfusion in mice].

Objective: To evaluate the neuroprotective effects of linagliptin, a dipeptidyl peptidase-4(DPP-4) inhibitor, on cerebral ischemia/reperfusion(I/R) injury in mice. Methods: BALB/c mice were randomly divided into Sham group, I/R group and linagliptin (2.5, 5 and 10 mg/kg) +I/R group, 8 mice in each group. The mice in the linagliptin group were administrated by gavage 3 weeks before I/R. I/R injury model was induced by MCAO, neurological deficit scores(n=8) and infarct volume(n=4) were assessed 24 h following reperfusion. Forty-eight hours following reperfusion, mice were euthanized, the contents of glutathione (GSH), malondialdehyde (MDA), phosphoinositide 3 kinase (PI3K), phosphoprotein kinase B (p-Akt) and rapamycin target protein (mTOR) in brain tissue were measured (n=4). Results: Compared with the I/R group, the neurological deficit score and infarct volume were significantly decreased in the linagliptin pretreatment group after 24 h reperfusion (P＜0.05); the MDA content in the brain was significantly decreased (P＜0.05), while the GSH, PI3K, p-Akt and mTOR levels were significantly increased (P＜0.05). Conclusion: This study proves that linagliptin exerted a neuroprotective effect in I/R mice, which may be mediated by activation of the PI3K/AKT/mTOR pathway.

miR-15a regulates oxygen glucose deprivation/reperfusion (OGD/R)-induced neuronal injury by targeting BDNF.

Multiple microRNAs (miRs) have also been implicated in ischemic brain injury. This research intended to probe the regulatory function and the mechanism of miR-15a on the ischemic brain injury induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in neurons of rats. The OGD/R model was established with the cortical neurons separated from rats. After transfection with miR-15a mimic negative control (NC), miR-15a mimic, miR-15a inhibitor NC and miR-15a inhibitor, the OGD/R-induced apoptosis were detected. Using bioinformatic softwares including TargetScan, miRanda, and miRWalk to predict the underlying targets of miR-15a, and the binding of miR-15a with brain-derived neurotrophic factor (BDNF) were validated with double-fluorescein reporter assay system. The expression levels of BDNF mRNA and protein were detected with qRT-PCR and western blot. The effect of miR-15a on PI3K/AKT pathway in neurons submitted to OGD/R was also investigated. The findings showed that miR-15a may mediate the apoptosis of neurons submitted to OGD/R, and lower expression of Bcl-2 and higher expression of Bax and cleaved caspase-3 were observed. BDNF was screened as the candidate target, and the direct binding of miR-15a with 3'-UTR of BDNF were verified. Further research showed that miR-15a downregulated the expression of BDNF mRNA and protein, thus exerted negative regulatory effect on the OGD/R injury. PI3K/AKT pathway may be related to the regulatory effect of miR-15a. Our findings contribute to uncovering novel pathogenesis for ischemic brain injury.

Phosphorylated mTORC1 represses autophagic-related mRNA translation in neurons exposed to ischemia-reperfusion injury.

OBJECTIVES: The sequential reactivation of mechanistic target of rapamycin (mTOR) inhibited autophagic flux in neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R), which was characterized by reduction of autophagosome formation and restriction of autolysosome degradation. However, its detailed molecular mechanism was still unknown. In this study, we further explore the existing form of mTOR and its suppression on the transcriptional levels of related mRNA from neurons exposed to ischemia-reperfusion injury. METHODS: The OGD/R or middle cerebral artery occlusion/reperfusion (MCAO/R)-treated neurons was used to simulate ischemia/reperfusion injury . Autophagy flux was monitored by means of microtubule-associated protein 1 light chain 3 (LC3) and p62. The reactivation of mTOR was determined by phosphorylation of ribosomal protein S6 kinase 1 (S6K1). Then the inhibitors of mTOR were used to confirm its existence form. Finally, the mRNA transcription levels were analyzed to observe the negative regulation of mTOR. RESULTS: The sequential phosphorylation of mTOR contributed to the neuronal autophagy flux blocking. mTOR was re-phosphorylated and existed as mTOR complex 1 (mTORC1), which was supported by phosphorylation of S6K1 at Thr 389 in neurons. In addition, the phosphorylation of S6K1 was decreased roughly by applying mTORC1 inhibitors, rapamycin and torin 1. However, the administration of mTORC1/2 inhibitor PP242 could recover the phosphorylation of S6K1, which suggested that mTORC2 was involved in the regulation of mTORC1 activity. In paralleling with reactivation of mTORC1, related mRNA transcription was repressed in neurons under ischemia-reperfusion exposure in vivo and in vitro. The mRNA expression levels of LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b were decreased in neurons after reperfusion, comparing with ischemia-treated neurons. CONCLUSIONS: The reactivated mTORC1 could suppress the transcription levels of related mRNA, such as LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b. The research will expand the horizons that mTOR would negatively regulate autophagy at transcription and post-translation levels in neurons suffering ischemia-reperfusion injury.

High-specificity double-stranded DNA detection with a "humanoid" molecular beacon and TALEs.

Owing to the limitation of DNA-specific recognition, improving the sensitivity of bacteria detection without additional culture poses a considerable challenge. Herein, a sensitive, novel, and high-specificity fluorescence detection method was developed using transcription activator-like effectors (TALEs) as the specifically recognized element. Based on the property of TALEs for recognizing double-stranded DNA, the detection method was first proposed. Its sensitivity was further improved by isothermal amplification of RPA. The "humanoid" molecular beacon (H-MB) was designed to achieve signal output. The steric hindrance of the protein opens the stem, enhancing the fluorescence intensity of an attached FAM/BHQ-1 pair. This method can be used to detect Escherichia coli O157:H7 with single-base mismatch specificity and high sensitivity (detection limit of 3 cfu mL-1) and can be applied in actual sample detection by boiling the sample for 10 min. TALEs can be assembled according to the target, so the method can be used to detect other foodborne pathogens and viruses in the food safety and clinical fields.

A new idea for visualization of lesions distribution in mammogram based on CPD registration method.

BACKGROUND: Mammography is currently the most effective technique for breast cancer. Lesions distribution can provide support for clinical diagnosis and epidemiological studies. OBJECTIVE: We presented a new idea to help radiologists study breast lesions distribution conveniently. We also developed an automatic tool based on this idea which could show visualization of lesions distribution in a standard mammogram. METHODS: Firstly, establishing a lesion database to study; then, extracting breast contours and match different women's mammograms to a standard mammogram; finally, showing the lesion distribution in the standard mammogram, and providing the distribution statistics. The crucial process of developing this tool was matching different women's mammograms correctly. We used a hybrid breast contour extraction method combined with coherent point drift method to match different women's mammograms. RESULTS: We tested our automatic tool by four mass datasets of 641 images. The distribution results shown by the tool were consistent with the results counted according to their reports and mammograms by manual. We also discussed the registration error that was less than 3.3 mm in average distance. CONCLUSIONS: The new idea is effective and the automatic tool can provide lesions distribution results which are consistent with radiologists simply and conveniently.

A target triggered proximity combination-based fluorescence sensing strategy for adenosine detection.

Adenosine is a potent physiological and pharmacological regulator, and its abnormal level is closely related to disease development. The sensitive and specific detection of adenosine is crucial for health evaluation and disease diagnosis. In this work, a target triggered proximity combination-based fluorescence sensing strategy is developed for the sensitive and specific detection of adenosine. A difunctional probe showing target recognition and signal amplification is designed, by integration of DNA linker-connected split aptamer fragments with a fragment-elongated polymerase/nicking template. The presence of adenosine would glue the split aptamers, which triggers the two distal aptamer fragments to combine with each other into proximity. The approaching aptamer fragment ends then initiate the strand displacement amplification (SDA) reaction, generating numerous DNA primers. The DNA primers further hybridize with a padlock probe and initiate the rolling circle amplification (RCA) reaction, producing numerous G-quadruplex sequences. The G-quadruplex sequences finally bind with Thioflavin T to obtain enhanced fluorescence signals. The method exhibits a linear correlation within the adenosine concentration range from 5.0 × 10-7 M to 2.0 × 10-5 M (R = 0.999) with a detection limit of 8.4 × 10-8 M, and a good selectivity to distinguish adenosine from its analogues. The recoveries of adenosine in human serum are from 91% to 94%, demonstrating that the system works well in biological fluids. The proposed sensing strategy is anticipated to hold promise in biochemical research, clinical diagnosis and disease treatment.

cPKCγ membrane translocation is involved in herkinorin­induced neuroprotection against cerebral ischemia/reperfusion injury in mice.

Herkinorin is an opiate analgesic with limited adverse effects, functioning as a primary selective atypical opioid µ agonist. The present study aimed to identify whether herkinorin has a positive effect on ischemic/reperfusion (I/R) injury. Adult male C57BL/6 mice were randomly divided into five groups: i) Naïve, ii) sham, iii) I/R, iv) I/R with dimethyl sulfoxide (I/R+D) and v) I/R with herkinorin (I/R+H). The I/R injury model was induced by occluding the middle cerebral artery for 1 h followed by 24 h or 7 days of reperfusion. Neurobehavioral scores and sensorimotor functions were examined 24 h and 7 days following reperfusion. In addition, infarct volumes were examined at these time points using a 2,3,5­triphenyltetrazolium chloride assay. Herkinorin treatment improved neurobehavioral and sensorimotor functional recovery from I/R­induced brain injury. There was a significant decrease in infarct volume in the I/R+H group at 24 h or 7 days following reperfusion compared with the I/R and I/R+D groups. Western blotting suggested that the decrease in conventional protein kinase C γ (cPKCγ) membrane translocation in the peri­infarct region may be attenuated by herkinorin pretreatment. These results indicated that herkinorin may be beneficial in I/R­induced mouse brain injury, and this may be attributed to the membrane translocation of cPKCγ following activation.

cPKCγ-Modulated Autophagy in Neurons Alleviates Ischemic Injury in Brain of Mice with Ischemic Stroke Through Akt-mTOR Pathway.

We have reported that neuron-specific conventional protein kinase C (cPKC)γ is involved in the development of cerebral hypoxic preconditioning (HPC) and the neuroprotection against ischemic injuries, but its molecular mechanism is unclear. In this study, the adult and postnatal 24 h C57BL/6J wild-type (cPKCγ+/+) and cPKCγ knockout (cPKCγ-/-) mice were respectively used to establish the models of middle cerebral artery occlusion (MCAO)-induced ischemic stroke in vivo and oxygen-glucose deprivation (OGD)-treated primarily cultured cortical neurons as cell ischemia in vitro. The results showed that cPKCγ knockout could increase the infarct volume and neuronal cell loss in the peri-infarct region, and enhance the neurological deficits, the impaired coordination, and the reduced muscle strength of mice following 1 h MCAO/1-7 days reperfusion. Meanwhile, cPKCγ knockout significantly increased the conversion of LC3-I to LC3-II and beclin-1 protein expression, and resulted in more reductions in P-Akt, P-mTOR, and P-S6 phosphorylation levels in the peri-infarct region of mice with ischemic stroke. The autophagy inhibitor BafA1 could enhance or reduce neuronal cell loss in the peri-infarct region of cPKCγ+/+ and cPKCγ-/- mice after ischemic stroke. In addition, cPKCγ knockout and restoration could aggravate or alleviate OGD-induced neuronal ischemic injury in vitro through Akt-mTOR pathway-mediated autophagy. These results suggested that cPKCγ-modulated neuron-specific autophagy improves the neurological outcome of mice following ischemic stroke through the Akt-mTOR pathway, providing a potential therapeutic target for ischemic stroke.

Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

Label-free nucleic acids detection based on DNA templated silver nanoclusters fluorescent probe.

Based on DNA templated Ag NCs (DNA/Ag NCs) fluorescent probe, a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1 (HIV-1) DNA. Firstly, a hairpin probe, containing target DNA recognition sequence and guanine-rich sequence, was designed to hybridize with the target DNA and form a blunt 3'-terminus DNA duplex. Then, exonuclease III (Exo III) was employed to stepwise hydrolyze the mononucleotides from formed blunt 3'-terminus DNA duplex, releasing the target DNA and guanine-rich sequence. Finally, DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence, leading to an enhanced fluorescence signal for detection. The proposed method could detect as low as 2.9×10(-10) mol L(-1) HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA. Furthermore, the method possessed perfect recoveries in cells lysate and human serum, showing potential to be used in biological samples.

Synthesis of polyethyleneimine capped carbon dots for preconcentration and slurry sampling analysis of trace chromium in environmental water samples.

Carbon dots capped with polyethyleneimine (CD-PEI) were synthesized and applied in selective separation and preconcentration of trace Cr(VI). Dispersed particle extraction (DPE) slurry sampling with flame atomic absorption spectrometry (FAAS) was used to selectively and sensitively determine Cr(VI) in water samples. The as-synthesized CD-PEI was confirmed by Fourier transform infrared spectroscopy, high-resolution transmission electron microscopy, elemental analysis, fluorescence and zeta potential measurement. The adsorption of Cr(VI) on CD-PEI was evaluated. Its isothermal adsorption was studied and fitted in the Langmuir model. Nearly 85% of Cr(VI) was adsorbed within 10 min showed that the CD-PEI exhibited fairly fast kinetics for the sorption of Cr(VI). Experimental conditions, including the content and size of CD-PEI, sample pH, adsorption time, sample volume, slurry volume and interfering ions, were further optimized to obtain efficient preconcentration and high-precision determination of Cr(VI). CD-PEI with small size turned to be a good candidate for the preparation of slurry. CD-PEI served not only as a promising adsorbent for separation and preconcentration of Cr, but also a signal-enhancing agent in FAAS. The method achieved an enhancement factor of 30 and a detection limit (S/N=3) of 0.21 µg L(-1) Cr(VI) with a consumption of 14.0 mL sample and an adsorption time of 5 min, which provided two times of signal enhancement. The RSD for 11 replicate measurements of 5.0 µg L(-1) Cr(VI) was 2.8%. The possible signal enhancement mechanism was proposed. The developed method has been applied to determine trace Cr(VI) in a variety of water samples.

Istradefylline, an adenosine A2A receptor antagonist, for patients with Parkinson's Disease: a meta-analysis.

OBJECTIVES: To assess the efficacy and safety of istradefylline as an adjunct to levodopa in patients with Parkinson's Disease (PD). METHODS: In this study, we searched the Cochrane Library, MEDLINE, Embase, China Academic Journal Full-text Database (CNKI), China Biomedical Literature Database (CBM), Chinese Scientific Journals Database (VIP), and Wanfang Database. The quality of included studies was strictly evaluated. Data analyses were performed by the Cochrane Collaboration's RevMan5.0 software. RESULTS: Five randomized controlled trials (RCTs) were included. The result showed a significant reduction of the awake time per day spent in the OFF state and improvement of the Unified Parkinson's Disease Rating Scale (UPDRS) Part III in the ON state when receiving istradefylline compared with patients receiving placebo. There was no significant difference between the istradefylline 20mg and the istradefylline 40 mg groups in the UPDRS Part III in the ON state (WMD=1.27, 95% CI [-0.40, 2.95]). The results showed significant differences in dyskinesia (RR=1.63, 95% CI [1.16, 2.29]) compared to istradefylline 40 mg with placebo. There was no significant statistical difference with regard to other adverse events. CONCLUSIONS: The present study showed that istradefylline is safe and effective as an adjunct to levodopa in patients with PD. Future large-scale, higher-quality, long-treatment, and placebo-controlled trials are needed.

[Correlation analysis between the PM2.5, PM10 which were taken in the hazy day and the number of outpatient about breathing sections, breathing sections of pediatrics in Shanghai].

In order to explore the correlation between the concentration of PM10, PM2.5 which were taken during the day of haze pollution and the average number of outpatient service, pediatrics. Based on the date with the number of pediatric outpatient and the concentration of PM2.5, PM10 which were taken form the haze days on 1 January 2009 - December 31 in 6 hospital in shanghai such as xinhuan hospital, we analyzed the data and executed the risk evaluation. The results showed that: in the haze day, when the average concentration of PM10 increase 50 microg/m3, the average number of the outpatient service and pediatric clinic increased 3% and 0.5%, when the average concentration of PM2.5 increase 34 microg/m3, the average number of the outpatient service and pediatric clinic increased 1.9% and 3.2%, Also, the pollution of PM10 and PM2.5 has a larger cumulative effects on the number of outpatient service. And the accumulation effect will be To maximize after 6 days behind the haze pollution. Thus, PM2.5, PM10 which were taken during the haze of pollution in shanghai, has certain influence on the average number of outpatient service, pediatric clinic.