| The carboxyl-terminal group Cys-Asn-Ser is the pharmacophore of anti-inflammatory peptide Met-Gln-Cys-Asn-Ser (MLIF). In this study, two new analogs (Tyr-Cys-Asn-Ser and His-Cys-Asn-Ser)of MLIF and the N-terminal of MLIF Cys-Asn-Ser were designd and synthesized by solid phase synthesis. The pharmacological activity and premerily mechanism on cerebral ischemia-reperfusion injury of the two analogs of MLIF were studied in comparing with the N-terminal of MLIF Cys-Asn-Ser.Methods1. In this study, two new analogs (Tyr-Cys-Asn-Ser and His-Cys-Asn-Ser) of MLIF and the N-terminal of MLIF Cys-Asn-Ser were designd and synthesized by solid phase synthesis. Sephadex G10 gel column was used for desalination treatment. HPLC and Mass Spectrometry were used to purify and identify the chemical structure of these peptides.2. Focal cerebral ischemia was induced in male SD (Sprague Dawley)rats using an intraluminal monofilament blockade of the MCA (Middle cerebral artery).After 1.5h of MCA occlusion, the animals were killed at 24h of reperfusion. In order to observe the effect of these peptides on cerebral ischemia-reperfusion injury, the neurological behavior were evaluated by Longa’s scoring method; Morphological changes were investigated by HE staining and the infarction size of cerebral tissue were measured by TTC staining technique at twenty-four hours after reperfusion.3. In order to study the mechanism of neuroprotective effect of these peptides, the level of oxidative stress markers (malondialdehyde, MDA and superoxide dismutase, SOD) were detected in brain tissue homogenate for evaluated the antioxidant activity. Moreover, MPO, TNF-αand IL-1βwere also measured in order to assess the anti-inflammatory activity of these peptides.Result1. The chemical structure of two new analogs (Tyr-Cys-Asn-Ser and His-Cys-Asn-Ser) of MLIF and the N-terminal of MLIF Cys-Asn-Ser were identified by HPLC and MS. The yields of them were 20.44%,33.95% and 41.53% respectively. The puritys of them are more than 90%.2. After MCAO, Ischemia-reperfusion group rats showed obvious neurological deficits; ischemic brain tissue exhibited a visible infarction; Morphological examination showed obvious ischemic damage. The neurological deficits were improved and the size of infarction was decreased after MCAO in rats which were pretreated and treated with these peptides. Moreover, the morphplogy of the occluded area were improved in different degree.The results showed that the pharmacological activity of MLIF new analogues has been increased compared with the small molecule peptides Cys-Asn-Ser.3. The MPO activity was obviously increased following 1.5h of ischemia and 24h reperfusion in comparig with the sham group. His-Cys-Asn-Ser, Cys-Asn-Ser and Tyr-Cys-Asn-Ser could obviously decrease the MPO activity and alleviate the neutrophil infiltration in ischemic brain tissue (P<0.05), especially in groupⅥ(His-Cys-Asn-Ser) (P＜0.01). IL-1βand TNF-a were increased in model group, Cys-Asn-Ser, Tyr-Cys-Asn-Ser and His-Cys-Asn-Ser could suppress the expression of IL-1βand TNF-a. The results showed that the anti-inflammatory activity of MLIF new analogues has been increased compared with the small molecule peptides Cys-Asn-Ser. Among them, the anti-inflammatory activity of His-Cys-Asn-Ser has been significantly enhanced4. After ischemic-reperfusion, the MDA levels was significantly increased in rat’s brain homogenate (P＜0.01). The MDA levels of each peptide group and Nimodipine treatment group were decreased significantly than model group (P<0.05). However, they had no obvious effect on the MDA levels in serum. In comparing with the sham group, the vitality of SOD in brain homogenate and serum of the model group were slightly decreased, the drug treatment groups showed increasing tendency, however, in comparing with the model group, there was no significant difference among them(P>0.05). Experimental results show that the N terminal His and Tyr of MLIF analogues have no effect on antioxidant activity for the small molecule peptide. ConclusionsBased on the above findings, the anti-inflammatory activity of Tyr-Cys-Asn-Ser and His-Cys-Asn-Ser has been strengthened, in comparing with the pharmacophore (Cys-Asn-Ser) of MLIF. Moreover, these peptides still have some antioxidant activity. Therefore, anti-inflammatory and antioxidant effect of them possible correlated with their protective effects on Cerebral Ischemia-Reperfusion Injury. |
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